The Secrets of Proteins

Popular Science THE SECRETS OF PROTEINS MEDHA S. RAJADHYAKSHA SUKANYA DATTA NATIONAL BOOK TRUST, INDIA ISBN 81-23...

Author: MEDHA S. RAJADHYAKSHA SUKANYA DATTA

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Science

THE SECRETS OF

PROTEINS MEDHA S. RAJADHYAKSHA SUKANYA DATTA

NATIONAL BOOK TRUST, INDIA

ISBN 81-237-3105-1 First Edition 2000 First Reprint 2006 (Saka 1927). © Medha S. Rajadhyaksha & Sukanya Datta, 2000 Published by the Director, National Book Trust India A-5 Green Park, New Delhi -110016

We dedicate this book with grateful thanks to Dr Bal Phondke who introduced us to the joys of popular science writing

Contents Acknowledgements

ix

Foreword

xi

Preface 1.

Introducing Proteins

xiii 1

2. Of Sequences, Shapes and Sizes

15

3. Proteins at Work

25

4. Making Proteins

46

5.

61

Editing Proteins

6. Commuting to Work

70

7. Studying Proteins

76

8.

89

Painful Proteins

9. Proteins—Food for Thought

97

Further Reading

115

Index

117

Acknowledgements This book owes its genesis to Dr Bal Phondke but for whom it would have remained just an idea. Thanks are also due to Dr Hema Subramaniam and Ms Radhika Kelkar for going through the manuscript. The staff at NBT, especially the artists who illustrated the book deserve our thanks. Manju Gupta merits special thanks for her patience, perseverance and meticulous follow-up but for which this book would not have seen the light of day. There are others who have perhaps not been named— to them all who made this book possible, our thanks.

Foreword This book, a nice blend of theoretical knowledge with practical implications, is obviously intended for both the educated general reader as well as the scientist well versed in the field of biochemistry and biology. The titles of the chapters give a summing up of the whole book. From chapter to chapter there is a step-bystep progression in knowledge of the structure, transport and function of proteins. Tables and line diagrams further help the reader in understanding the secrets of proteins. The last chapter is of more direct practical application to the unfortunate disorder prevalent in children, namely, Protein Energy Malnutrition in several parts of the world, including India. The world is now becoming aware that this condition results as much from deficiency of food, as from lack of understanding of the role of various dietary items, especially proteins. The book is both knowledgeable and enjoyable to read and I congratulate both Dr Medha Rajadhyaksha and Dr Sukanya Datta in achieving this dual purpose. Bombay Hospital Mumbai

Prof. DARAB K. DASTUR

Director

Preface It is only a little twig With a green bud at the end; But if you plant it, and water it, And set it where the sun will be above it, It will grow into a tall bush With many flowers, And leaves which thrust hither and thither sparkling. . . But if you take my twig, And throw it into a closet With mousetraps and blunt tools, It will shrivel and waste. And, some day, When you open the door, You will think it an old twisted nail, And sweep it into the dustbin With other rubbish. — Amy Lowell This book too is a little twig with a bud; a small introduction to proteins, their synthesis and regulation. Whatever little is said here is not the complete story, but it may help new questions to rise in young minds. That the twig might take root as interest in molecular biology is a gratifying thought.

1

INTRODUCING PROTEINS Problems worthy of attack, Prove their worth by hitting back. — Piet Hein, Danish scientist The problem of food and nutrition is one such problem. It has been a central issue in the past and continues to be one in the present century. With each technological advance we feel we are close to solving it, but it defies solution at every step especially as population pressure mounts all over the world. Today, human populations and resources are distributed unevenly over the world. Some of us panic over problems related to malnutrition while others worry about the health hazards of overeating. Types of foods and their nutritive value are of concern to the privileged and ironically more so to the underprivileged. Our concern for foods that we should or should not eat has given us a fair idea about what a balanced diet should be. Even a schoolgoing child will rattle off that a proper diet should include proteins, carbohydrates, fats, minerals and vitamins. However, despite many years of study the last word is yet to be pronounced. A lot of available information related to a correct diet is ambiguous. Which foods prove to be best suited for ideal growth and development? In what quantities should they be taken? What should one eat to ensure a feeling of wellbeing? For an active and healthy long life what should be the pattern of consumption and why? Do different lifestyles call for different diet regimes? A lot of issues relating

2

T H E S E C R E T S O F PROTEINS

to food and nutrition are debatable. Not only is the common man's experience with recommended health foods variable but scientists too, have yet to come up with definite answers. A couple of decades ago, problems, especially of the Third World countries, zeroed down to deficiency of proteins in the diet. However, even as the United Nations took well-organised measures to meet the world protein requirements, the nutritive value of proteins itself was challenged. All the same, it is an undeniable fact that proteins are the most impressive of macro molecules. Proteins are the building blocks of our body and give cells their size and shape. The human body has hundreds and thousands of types of proteins. A number of them are yet to be discovered. Proteins are present in all sizes and shapes. They perform a variety of functions. An idea of their importance can be obtained from the fact that 65 per cent of the body weight is accounted for by water, while proteins make up one half of the rest. It is now believed that half the dry weight and 20 per cent of the total weight in an adult is protein. About half the body's protein is found in muscles, a fifth in bones and cartilage and a tenth in skin. The rest is distributed in other tissues. Most cellular processes are protein based. Proteins are little machines running the process of life. They support all mechanical work done in the cell. They are in charge of cellular communications; several hormones as well as their receptors are proteins. As versatile enzymes, they perform the uphill task of running the body chemistry. Closely associated with the heredity molecules, proteins act as regulators of all that goes on in the cell. Some proteins are common to most organisms, for they are indispensable to life itself. Such proteins are of special interest, for they indicate a sort of common cellular heritage reaching back to times far distant on an evolutionary scale. Such proteins have been used by scientists as evolutionary clocks ticking within us. They trace our evolutionary history. This astounding variety of functions ma' es

I N T R O D U C I N G PROTEINS 17

proteins, literally and figuratively, the most favoured foods for body as well as for thought. No wonder then, that the study of proteins has kept chemists, biologists, molecular biologists and computer scientists busy for years! Fascination with food dates back to ancient times. Some of the questions that remain pertinent even today were raised way back in the 16th century. About 200 years ago, though little was known about chemistry, physics or biology, folks cared to ask and answer questions with remarkable ingenuity. One such seeker of truth was the Italian scientist Santorio at the University of Padua. At a time when sophisticated techniques or equipments were unheard of, he devised thought-provoking experiments that in retrospect seem ridiculously simple. He carefully measured all that he ate and all that he excreted every day. Based on detailed observations, he reported that on an average his daily intake was 8 lbs (4 kg) while his output was only about 3 lbs (1.5 kg). As his weight remained steady, he wondered where the 5 lbs (2.3 kg) of his food 'disappeared' every day. It was only in 1637 that Rene Descartes suggested that nutrients circulating in blood 'distilled' into 'stuff' of animal tissue. It was also realised that plant food was being 'animalised'. Such pioneering work laid the foundation of the science of nutrition. Some of the earliest work on proteins was inadvertently reported by Jacopo Beccari, working at the University of Bologna. He argued: "Our bodies must, presumably, be composed of the same substance which serves as our nourishment." And he looked for a substance as 'gluey' as egg albumin and muscle protein in wheat flour. He did find the 'animal glue' he was looking for: it was the 'wheat gluten'. Oblivious to the fact that he had done so, Beccari had reported the first vegetable protein. Of course, the term 'protein' was not coined till 1838, i.e. several decades after Beccari's discovery but the story behind the term 'protein' is an interesting one and worth telling! Gerrit Mulder was a Dutch physician deeply involved

4

THE SECRETS OF PROTEINS

in chemistry. He worked with materials such as egg albumin, serum fractions and wheat gluten. By chemically modifying these substances from different origins by reacting them with acids, he purified a chemical that appeared to be common to all of them. After a detailed analysis he concluded that the common ingredient was made up of 40 atoms of carbon, 62 atoms of hydrogen, 10 atoms of nitrogen and 12 atoms of oxygen. Mulder sent his findings to Berzelius, the famous chemist of his time. Struck by the originality of the work, Berzelius wrote back in appreciation: "The name 'protein' that I am proposing for the organic oxide of fibrin and albumin, I chose to derive from the Greek word proteus, because it appears to be fundamental or primary substance of animal nutrition which plants prepare for herbivores and who in turn supply the carnivores." Mulder gracefully accepted the suggestion and the term 'proteins' came into usage. Interestingly, the Greek god Proteus was thought to have the ability to change shapes. True to name, the proteins turned out to be molecules that exist in Nature in a wide range of shapes and sizes. Literally, the Greek word proteios means 'primary or holding the first place'. Berzelius would not have known it then, but proteins have certainly justified and lived up to their name! No matter what their shape and size may be, these complex molecules of life are, as Mulder found, made up of carbon, oxygen, hydrogen and nitrogen. These four elements link up with other elements to form the structures of the living world. A number of these elements can be picked up from the atmosphere by cellular biochemical processes. Picking up nitrogen, however, is a problem. We have no mechanism by which we can convert atmospheric nitrogen into compounds we can use. As all proteins contain nitrogen, we can hardly do without them. Moreover, the other major macro molecules, the nucleic acids, too contain nitrogen. The cellular demand for nitrogen is substantial and real! The problem would have been

INTRODUCING PROTEINS

5

insurmountable, had it not been for Nature's generosity. Some organisms, which can convert nitrogen into the chemical form that we can use, are around to help out. Very small in size, each a single cell living in the soil, these are little machines that convert atmospheric nitrogen into compounds that enrich the soil. These are the so-called nitrogen-fixing bacteria. While some other groups of life forms also fix nitrogen, these bacteria do so with remarkable efficiency. It is these organisms that we depend on for nitrogen which our bodies so desperately need. The plants take up the nitrogenous compounds produced by bacteria. Once this is achieved, nitrogen enters the food cycle and becomes available to all living beings. It is a sobering thought that these almost invisible bacterial cells have been supporting the plant and animal world ever since its origin. It has taken us a long time to understand this relationship in terms of its chemistry. The more we have explored, the more we have discovered our acute dependence on other living beings on earth. Not only can we not fix atmospheric nitrogen, we cannot by ourselves make all the parts of proteins even if the raw material is provided. These ingredients must be an 'essential' part of our diet. In other words, we depend on outside sources to be able to make the proteins we need. Cooked, chewed and gulped, the proteins in our diet reach the intestine in a semi-fluid state. Here they are 'digested'. This is essentially a process of stepwise chopping of proteins into smaller units that can get into the bloodstream and reach every organ. These small units that the large dietary proteins get broken into are the 'amino acids'. These monomers, the amino acids, are the building blocks of proteins. They were of special interest to the early protein chemists. In order to understand what really made the protein molecules work it was imperative to understand the amino acids first. To produce amino acids for experimentation it was obligatory to break down proteins. In other words, it was necessary to recapitulate the

6


digestive process in a test-tube. Luckily, a mild treatment of the proteins with acid or alkali did the trick. As water molecules were involved in the reaction, proteins were said to be 'hydrolysed' to amino acids. The simplest amino acid glycine was first reported by the French chemist H. Braconnot as having been produced by acid hydrolysis of gelatin. Soon several other amino acids were discovered. Breaking down proteins to amino acids simplified matters for protein chemists. Now the unwieldy, incredibly large protein molecules could be understood as polymers of amino acids. The molecule of milk protein, lactoglobin, containing approximately 1864 carbon, 3012 hydrogen, 576 oxygen, 468 nitrogen and 21 sulphur atoms seemed much less formidable when seen as a combination of 20 small amino acid molecules. As the name suggests, the amino acids are acidic. And all of them also carry an amino group. An amino group is the scientific term for a chemical group made of hydrogen, carbon, oxygen and nitrogen combined in a specific proportion to form a characteristic structure. But this is not all what amino acids are. They have an additional chemical part that gives them their identity. This extra part may be just a molecule of hydrogen (as in glycine) or an elaborate little chain of carbon and hydrogen (as in valine, leucine and isoleucine). It may as well be another acidic group (as in glutamic or aspartic acid). Some molecules may have a COO' +NH3

I

€

H

R Fig. 1.1.a: This is the general formula for an amino acid along with pictures of amino acid. R represents a variable grouping of atoms.


7

ring structure as the side chain (as in tryptophan or histidine) while still others contain sulphur (as in cysteine and methioine). All the three groups: the acidic, the amino and the special characteristic group are held together by a single atom of carbon (Fig.l.l.a). It is the nature of the amino acid radical that distinguishes one amino acid from another. Proteins are made up by permutation and combination of 20 different amino acids. However, not all 20 amino acids need necessarily associate to form a given protein. Combination of varying numbers of amino acids in different sequences form the infinitely large variety of proteins. This is how an infinite number of proteins can be produced using only 20 building blocks (Fig.l.l.b). Scientists in the early part of the century struggled to work out the amino acid composition of a protein. The biochemists had two major technical problems. The first was to get intact proteins from the cells and that too in sufficient quantity. Proteins are delicate molecules, prone to degradation with a slight change in temperature or in the ionic conditions of the medium in which they are extracted. J.H. Northrop, M. Kunitz, E.T. Cohen and C.F. Cori from the USA along with other biochemists worked for over a decade to evolve the technique of separating intact protein molecules. The Swedish chemist A.V. Svedberg evolved an elegant technique of estimating the molecular weight of proteins. Suspended in a solution, proteins could be swirled in a centrifuge and subjected to a strong force that sedimented the proteins. Larger and heavier protein molecules sedimented faster than the smaller, lighter ones. The rate of their sedimentation gave a fair idea about the molecular size and weight of the proteins. This ingenious method was not only used to estimate the molecular features of protein molecules but a modified version could be used to separate out the proteins. The second problem that the biochemists faced was how to separate out the amino acids obtained after

8


ft

Serine

Glycine

Cysteine

Alanine ,

Proline

Valine

Fig. l . l . b : Amino acid structures.


Aspartic acid

Asparagine

Glutamic acid

Threonine

Leucine Fig. 1.1.b: Amino acid structures.

9

10


Glutamine

Isoleucine

Histidine

Lysine

Methionine Fig. l . l . b : Amino acid structures.


Phenylalanine

Tyrosine

Arginine

Tryptophan

Fig. l . l . b : A m i n o acid structures.

11

12


hydrolysis of proteins. A very simple technique developed by a Russian scientist, M.Tswett, in 1906, helped overcome the problem. Tswett was working with the photosynthetic pigments (chlorophylls) from plants. He separated the pigments on their differential ability to be adsorbed onto an inert material. The technique was called 'chromatography' as it was used to separate coloured substances. The potential of this technique was appreciated years later by the protein chemists A.T.R Martin and R.L.M. Synge who used for the first time a modified version of chromatography to separate amino acids. The method was a great success and its sophisticated edition is used today for analysis of amino acids. Amino acids can be classified into two broad categories. Under the first come those amino acids that our bodies can make. The second category of amino acids includes those that we must get readymade through all that we eat. We cannot make nine of them at a rate fast enough to support our needs. These amino acids are— leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine and histidine. They are called 'essential' for they have to be included in the diet. The term 'essential' merely refers to our dietary requirements. It does not mean that they have a more important role than the amino acids we can synthesize ourselves. It just means that since the body cannot make up for the deficiency in these amino acids, our diet must include these in adequate amounts. Once in our cells, these amino acids reorganise into proteins. Each one of them can make bonds with the other. The nature of this bond was described independently by Emil Fisher and Franz Hofmeister in 1902. They called this bond the 'peptide linkage'. The carboxyl or the acid group of one amino acid and the amino group of the other come together and react to form the peptide bond. Like a person holding out hands on either side and becoming a part of a chain, the amino acids participate in a peptide bond


13

linkage and can form chains of amino acids. Acid hydrolysis or hydrolysis during digestion of proteins involves breaking of this bond and reintroduction of the water molecule. A peptide made up of two amino acid is called a 'dipeptide'. When three amino acids are involved, the term used is 'tripeptide' and when many more are linked, then the term used is 'polypeptide'. All our proteins are essentially peptides. The 20 amino acids can join in all possible combinations to form short or long peptides (Fig.l.2)..In all peptides, the amino acid at one end has an amino group free and this end of the protein is called the 'amino end'. The other end is called the 'carboxyl end': the last amino acid has the carboxyl group free. The sequence of amino acids in a protein molecule is important: it determines the way proteins look and the work they do.

H

R1

H

N—C—C—rOH H

H O

R2 N—O-C—OH-

H

H O H

R1

H

H O

R2

H H O

Fig. 1.2: Example of peptide bond; R 1 and R 2 represent the side chains of two amino acids.

Identifying, isolating and working out the structure of all these amino acids has been a tricky job. Still more difficult, yet perhaps more exciting too, has been the discovery of the process by which amino acids are put in place by our cells to remake the proteins in our cells.

14


Understanding this puzzle of new protein formation is an important collective contribution of our century. Everyone who has been working on this problem has painstakingly put some irregular pieces of the jigsaw puzzle in place. How this has been done is a fascinating but long story. Now that we have a fair picture of how proteins are made, it is possible to take short cuts. Maybe, it is possible to redivide the picture into pieces that can be put back simply. Maybe, if we answer some questions, the story that has taken 100 years to be pieced together can be unfolded in the next few pages. For example, one can begin by asking: What do proteins look like and how do the amino acids link up to form the large protein molecule? As cells need proteins of all shapes and sizes, the molecules are necessarily tailor-made; then how is this done? Once made the proteins are put to their work. What are the things proteins can do in the cell? What happens if proteins are wrongly made or not made at all? Each of these questions when answered makes way for more. Each question hits back—and that is what makes them worth tackling!

29

OF SEQUENCES, SHAPES AND SIZES Twas brillig, and the slithy toves Did gyre and gimble in the wabe All mimsy were the borogoves, And the mome raths outgrabe. In the book Through the Looking Glass by Lewis Carrol, Alice reads this poem. She can make no sense of it till she is told how the words can be made to mean more by fusing and mixing. The same seems to hold true of the protein structure. The sequences of amino acids make no sense unless scientists work out how they form a structure that can do work in a cell. Deciphering the sequence of amino acids in a protein molecule was the first formidable task scientists tackled. And the next task was to work out the three-dimensional structure of the protein. These two protein problems seemed most crucial to biochemists. Understanding the structure and function of proteins—the molecules of life—appeared to be the most important mystery to be solved in the earlier part of the 20th century. There was a lot of excitement in the air. Groups worked at a frenzied pace, in a spirit of friendly rivalry, attempting to beat each other at getting to the answers. A number of techniques were discovered, refined and used ingeniously. Solutions to the two problems came almost simultaneously and most Nobel Prizes of that time were bagged by the scientists working on proteins! Working out the sequence of amino acids in a protein molecule is a formidable task. The procedures adopted at

16

T H E SECRETS OF PROTEINS

present by protein chemists are based on pioneering experiments done by Frederick Sanger and his colleagues at Cambridge University. Sanger dedicated 10 long years to a single molecule, insulin. Sanger choose the molecule for his study after careful thought. A protein hormone of considerable clinical importance, insulin is rather small in size. It was available in large quantities and was known to be made up of 51 amino acids. With the use of chromatography, it had been demonstrated that insulin had only 17 of the 20 known amino acids. The unresolved problem was to deduce the exact sequence of these amino acids in a molecule of insulin. Sanger developed an elegant method to work out the sequence of amino acids in the insulin molecule. He chopped up the molecule into fragments, analysed them and reconstituted the protein just like a linear jigsaw puzzle. The technique that proved to be most useful in separating amino acids was a modification of chromatography. Developed by Archer Martin and Richard Synge (for which they were awarded the Nobel Prize in 1952) this technique, called 'partition chromatography' could pick up individual amino acids on a chromatogram. i Sanger and his group began with hydrolysing the insulin molecule to small peptides, mostly tri and di peptides., The sequence of each bit was worked out by tagging the amino end of the peptide with a coloured substance. With the help of partition chromatography the coloured amino acid could be identified. Sanger and his group analysed several such peptides, each produced by cutting the insulin chain at a different point, using acid and a host of enzymes. Bit by bit, deducing the overlaps in the fragments, they got the exact sequence of amino acids in the insulin molecule. The molecule turned out to be made up of two chains, one with 21 amino acids and the other with 30 amino acids held together with sulphur-containing bonds. Sanger and his group published their work in 1953. Honoured

O F S E Q U E N C E S , S H A P E S A N D SIZES

17

with the Nobel Prize in 1958, Sanger opened the doors to solving the first of the protein puzzles. The second part of the puzzle was equally tricky. Work on three-dimensional structures of macro molecules had begun even before the sequencing of proteins was undertaken. This was because an elegant technique used for studying molecular structure had been discovered in the earlier part of the century. Still in use, the technique is called 'X-ray crystallography'. It was first used in 1912, by Lawrence Braggs to visualise the three-dimensional structure of crystals of common salt, sodium chloride. Some years later it was realised that the technique was the best one available to study three-dimensional protein structures. The X-ray diffraction technique records the pattern of rays diffracted from a purified crystal of protein or any other molecule that is exposed to X-rays. When a protein crystal is rotated, as X-rays pass through it, the atomic arrangement in the crystal causes diffraction of the rays. The pattern of diffraction can be recorded on a photographic plate as dark and light regions. This pattern can be analysed to deduce the three-dimensional coordinates of each atom in the molecule. The technique, though requiring a specialised and sophisticated analysis, attracted a number of scientists; and the 1951 May issue of the journal, Proceedings of National Academy of Sciences, carried seven independent papers reporting patterns produced by proteins of hair, feather, muscle, silk, horn, tendons and also other proteins. Interestingly, all these papers came from research laboratories of a single group led by Linus Pauling. One major approach to the study of protein structure was, and still is, the theoretical consideration of the nature of bonds that can be formed in a macro molecule. Linus Pauling had a strong grounding in these aspects and had published a classic book, The Nature of Chemical Bonds in 1939. Using his theoretical wisdom to understand

18

T H E SECRETS O F PROTEINS

diffraction, patterns obtained by X-ray crystallography, Pauling proposed a generalised model for fibrous proteins. He said that proteins had an 'alpha helical' structure. X-ray crystallography was used to study globular proteins as well. Max Perutz and John Kendrew had joined Lawrence Bragg at Cambridge and formed an excellent team devoted to protein structure analysis. Perutz worked on the haemoglobin, the oxygen-carrying protein in blood while Kendrew chose myoglobin, a smaller but related molecule, to work on. Kendrew published the detailed structure of myoglobin in 1958, closely followed by Perutz who published the haemoglobin structure in 1959. Both of them shared the Nobel Prize in 1962, for cracking the three-dimensional structure of the two globular proteins. Years of work, painstakingly done, brought rewards to them and widened the horizons of others who followed! With the two main protein problems effectively tackled, slowly and steadily a fair picture of the several proteins in our body emerged. We know a lot about protein structure and function now. Each protein in the cell has an amino acid sequence, a shape and size of its own. The 20 amino acids that are necessary for building the protein are all chemically different. The behaviour of a protein depends on the number and the type of amino acids that constitute it. It is also determined by the sequence in which these amino acids are linked. However, how a protein looks and behaves may change not only with a change in its amino acid sequence but also with the change in other molecules surrounding it. This makes protein molecules extremely versatile and helps them perform an amazing number of jobs in the cell. A cell can use the set of 20 amino acids to make its proteins. This is not such a limited option as it may appear. As there is total freedom to choose the sequence of these amino acids an enormous number of proteins can be generated. For example, let us try making a protein only


19

two-amino-acids long. Out of 20 amino acids any one can occupy any of the two places. With the first amino acid in the first position we can make 20 proteins with any of the other amino acids in the second position. We can make another 20 by putting the second amino acid in the first position, and another 20 by putting a third one in the first position. If we add on the number of possibilities by putting each of the 20 amino acids in the first position we would get 20 x 20 or 400 hundred possibilities. Most proteins in the cell are much longer. A huge number of proteins can at least theoretically be made. However, we make relatively few possible combinations of amino acids. This is because not all combinations fold well. Others do not remain folded for long. In addition, only a few combinations work suitably in the chemical milieu in the cell. Structurally stable and functionally useful proteins that can be made seem to be rather limited. This is because each protein has a special function in the body which it can perform successfully by having a unique design. Any change in the sequence often has drastic effects on the functional ability of the protein. More so because a change in amino acid sequence often results in the change of its shape. Suppose we were to take a long chain of beads and play around with it to see into how many shapes and sizes it can be folded. Doing this manually can be fun but time consuming. This can be done efficiently instead, on a computer. Further, we can instruct the computer what each bead looks like, what electrical charges it carries and how it behaves when other beads are nearby. Now the computer has to work a little more and come up with shapes that the chain could possibly make according to the rules specified. Some contours may be ruled out because the beads may have some special attractions or repulsions to certain other beads. Eventually, the computer would generate all viable shapes that take into account all possible constraints. An experiment of this type is worthwhile

20


because it helps us to predict how long-chained structures are formed. If we consider amino acids to be represented by beads, each with a special feature, the protein structure can be somewhat understood. Computer modelling of proteins has turned out to be an extremely useful method of finding the three-dimensional structure of a protein, especially as scientists are taking molecular short cuts to discover new proteins in our bodies. As each protein has its own set of amino acids linked in a chain, it is distinctive (Fig. 2.1). However, folding patterns of the peptide-chain are not always distinct. Certain patterns of peptide chain folding are commonly encountered. One pattern of folding that is seen very often is called the 'beta sheet'. The other is called the 'alpha helix'. If we take a strip of paper and fold and refold it as if to make a streamer and then open it up, we see what a beta (fi) sheet looks like. The protein fibroin, found in silk, looks like this. If we take the paper strip and wind it around our finger clockwise before releasing the paper, we get the structure called the 'alpha helix'. This is the structure described by Linus Pauling. The protein keratin present in our nails has a structure like this. Some proteins have uncommon shapes. Many proteins comprise of a mixture of beta sheets and alpha helices. Structures that are globular in shape are formed out of 6 sheets and a helices held together closely. Each such region is called a 'domain'. A domain is associated with a particular function. A protein may have several domains or just one. Small proteins have one domain while large, ones have many. When more than one domain is present in a protein, there is often a small chain of amino acids that joins them. In a protein several domains may come together in three dimension by a fold in the chain, linking them to form a shape that is very important for its function. Some proteins are not made up of just a single polypeptide chain. They are made of many chains held together to form a giant molecule. In some proteins the chains are

21

60

80

a-helix

100

Extended chain

OF SEQUENCES, SHAPES AND SIZES

a>> •uJ3 •s •S3 x) Si (0 M QJ

1 Iin .s•8 O & OH t-H

(S

20 nm

Sphere

p-sheet

40

60 £

22

T H E SECRETS O F P R O T E I N S

loosely associated to form a stable, functional structure. The protein collagen is made of three polypeptides wound around each other. In some proteins, such loose binding of chains is not sufficient to ensure stability. A firm chemical bond holds such polypeptides together. The two chains of amino acids in insulin, for example, are held in place by strong chemical bonds. At times, small proteins made up of single polypeptides join to form a long chain of protein. This is called polymerisation. Each of the small proteins is a subunit or 'monomer' that can do little by itself but together with other subunits works efficiently. Such subunits may form sheets and tubes. The protein that gives a cell a typical shape is tubulin. It forms the micro filaments which constitute the micro skeleton within the cell. Monomers of tubulin polymerise to form a tubular assembly. Some proteins can form a spherical assembly by arranging monomers in a typical array The variety of shapes and sizes that the protein molecule can form is remarkable. A correct shape with the right contours must be attained by all proteins if they are to participate in the cellular machinery. Folding ensures structural stability. It also exposes certain sites or domains that participate in a reaction selectively (Fig.2.2). Technically, the structure of a protein molecule can be described taking into account all its complexity at different levels. The term 'primary structure' refers to the sequence of all amino acids that make up the polypeptide chain. The 'secondary structure' refers to the extended or helical conformation of the peptide; the 'tertiary structure' refers to a further folding that gives compactness to the molecule; while the 'quaternary' structure refers to the arrangement of all constituent polypeptides in space. Proteins having more than one polypeptide chain are called 'oligomeric proteins' and their individual chains are called 'protomers'. Once made and folded, the proteins have certain areas where other molecules cannot easily reach. Other areas of


23

PRIMARY

Amino acid sequence SECONDARY

a-helix

TERTIARY

Coiling

QUATERNARY

Combination of chains

Fig. 2.2: Protein molecules must fold to the correct configuration to function.

24


the folded protein form its surface and are exposed to the molecular surroundings. The chemical groups that stay exposed are the ones that will essentially decide the reactions in which the protein molecule will participate. Although the unexposed region of the protein does not directly participate in a reaction it does have a say in the matter. It helps in giving the right contours to the reactive groups. A change in conformation of a protein changes the way it behaves. The looks of a protein are most important for it to work well. Firstly, the number and sequence of amino acids and its 'primary' structure must be properly made. This should fold correctly to form the beta sheets or alpha helices, the secondary features of the molecule. It must further form domains and the final three-dimensional structure with correct reactive groups in position; eventually it must assemble in the right place. The cell machinery is geared to take care of all these features. It attends to all the molecular details with unbelievable precision. And how it does this is the important part of the puzzle that a lot of scientists have worked very hard to solve.

3 PROTEINS AT WORK I'm a business and a factory, the workforce and the boss; I'm the brains and the belly And I never make loss. — William Scanmell These lines, originally describing a dinosaur, also aptly describe the rather monopolistic role that proteins play in the cell. They occupy all the possible locations and positions in the cell. Although deoxyribo nucleic acid (DNA) has garnered a lot of publicity as the 'master molecule', there is little it can do without the behind-the-screen support of proteins. DNA can neither replicate nor express without proteins. The cellular architects are proteins. The work force are proteins. In a fine play of diverse capabilities, molecules that build are proteins and the ones that destroy are proteins too. It is incredible that a single type of molecule can carry out so many diverse and at times, opposing functions in the cell. How this seeming paradox is handled by proteins merits a closer look. Proteins give form to the cell. Apparently the inside of the cell seems to be filled with a jelly-like matter called the 'cytoplasm'. However, on closer look a 'skeleton' of protein fibres that serves as molecular scaffolding holding the cytoplasm, can be seen. The cellular skeleton or 'cytoskeleton' as it is known, plays an important role in the dynamic processes that the cell undergoes in its

26


Plasma membrane Microtubule doublet

Microtubule doublet

Radial spoke Dynein arms

PROTEINS AT WORK

27

Fig. 3.1.a & b : Cilia and eukaryotic flagella have nine microtubule doublets surrounding a central pair of microtubules.

28


lifetime. Cells come in many shapes and sizes. Some are ovoid like the red blood cells while some are polygonal liver cells. Nerve cells have a complex shape while muscle cells are spindle shaped. Once formed, most cells hold on to their respective shapes. Some cells, like some types of white blood cells, change their shapes when required. Invariably all cells acquire their typical threedimensional features because of the elaborate protein— cytoskeleton. The cytoskeleton is made of three classes of protein fibres. These are the actin filaments, microtubules and intermediate filaments. In addition, there are other accessory proteins that connect these fibres to each other, to other cell structure or to the cell membrane (Fig. 3.1). Actin filaments are made up of the protein actin. This protein is related to but distinct from the actin found in the muscle cells. An actin filament consists of two chains helically twisted around each other. The major function of actin filament is to exert force, especially when a cell moves or changes its shape. Microtubules are a network of fibres that crisscross the cytoplasm. The microtubules are made of two closely related polypeptides called 'alpha tubulins' and 'beta tubulins'. These form the protofilaments. Thirteen protofilaments together form a fibre of microtubule. Microtubules are continuously made and dismantled as and when needed in the cell. Accessory proteins called the 'microtubule-associated proteins' influence the state of equilibrium between the breaking down and reassembly of microtubules. Intermediate filaments fall into five general classes and each found in a particular type of cell are engaged in specialised functions. For example, intermediate filaments in the skin cells are made up of the protein 'keratin', while those in the muscle are made up of 'desmins'.

P R O T E I N S AT W O R K

29

All three varieties of protein fibres that form the cytoskeleton found in varying quantity in the cells, give cells an interior structure and an overall dimension. The membrane of the cell has proteins that act as anchors to these cytoskeletal proteins. A change in cell shape often involves alterations in anchor proteins. Proteins also cement cells together. Known as adhesion molecules, some proteins displayed on the cell surface not only act as a glue but also help cells interact with other cells. The cell, as a unit of our body, is often likened to a brick that is used to build a house. However, there is one fundamental difference between the two. The cell is not merely a structural module of the body; it is a functional unit as well. A cell carries out myriad functions—some routine and mundane, others highly remarkable and specialised. Every cell needs energy to survive and carry out its metabolic activities. To provide this energy many biochemical reactions take place in the cell. Without timely help from some specialised proteins, the enzymes, these reactions would proceed at snail's pace. Most chemical reactions involve breaking up of molecules or joining them. Left to themselves these reactions would take place very slowly. Moreover, as all reactions are reversible, they would reach a point where the products and reactants would be present in equilibrium. The products formed would reform the reactants immediately. The reaction would not proceed effectively in any particular direction. In case of such a chemical stalemate the cell machinery would grind to a halt. However, thanks to the enzymes, this cellular disaster never happens. Enzymes control the rate of reactions. They can accelerate the rate of a chemical reaction a millionfold. Their stupendous catalytic powers are attributed to their capacity to bind to the reacting molecules in a precise configuration and their ability to make or break chemical bonds at ambient cellular conditions. Enzymes hasten the reactions,

30


not only by bringing the reacting molecules close to each other but also give the reaction a direction by helping to stabilise the intermediate molecules. Enzymes help drive the reaction in one direction, either forward or backward, as may be necessary. All enzymes are essentially proteins. That makes proteins indispensable for all metabolism in a cell! • In the cell one reaction often follows the other almost immediately Such reactions are helped by a group of proteins that assemble to form the multi-enzyme complex. In terms of biological function, enzymes are the largest class of proteins. Over a thousand different enzymes are known and each catalyses a different kind of chemical reaction. Even in case of multi-enzyme complexes, the variety of cellular sites where they abound is stunningly high. Many proteins that are assembled in the mitochondria are such complexes. These are involved in reactions that generate energy-rich molecules and are indispensable for cell survival. Proteins, as enzymes, have a wide range of functions. Some that are packed into lysosomes are the ones that chop up molecules and are known as 'lytic' enzymes. A large number of enzymes are present in the nucleus, some of them specially helping the DNA to form a copy of itself and some helping the transcription of the messages for protein synthesis. Physiologically important enzymes are present in the cytoplasm too. Some of them help in breaking down the sugars. All cell metabolism, no matter which compartment of the cell it is taking place in, is governed by proteins acting as enzymes. All cellular, metabolic pathways are controlled by enzymes either acting simultaneously, or acting one after another in different stages of the pathway. The interesting point to be noted is that although the entire biochemistry of a metabolic reaction may be a complex one, each step is a simple chemical reaction catalysed by a unique protein— the specific enzyme!


31

Proteins act as communicators. They carry messages from one part of the body to another. Some hormones, the chemical messengers that travel through the body and help in a number of our body functions, are proteins. However, not all hormones are proteins. Some are a separate class of chemicals called 'steroids'. Whether proteins or steroids, hormones are potent messengers made by glandular tissues called the 'endocrine glands'. Hormones exert their influence on an organ or tissue that is present away from the site at which they are manufactured. In response to the messages conveyed by the hormones, the responding tissues modulate their metabolism. The hormone insulin, for example, is synthesized by the pancreas. Secreted into the blood, it travels to various tissues and has profound influence on carbohydrate metabolism. Over the years, a large number of protein hormones have been studied and their mode of action has been elucidated at the molecular level. Study of protein hormones dominates the science of endocrinology and is a story in itself! There are also protein messengers that do not travel through the bloodstream but diffuse through the tissue and act locally. All these messenger molecules, whatever may be their mode of dissemination, are received by the cell on which they act, with the help of another set of proteins called the 'receptors'. The receptor proteins may be present on the cell membrane or may be dissolved in the cytoplasm. True to their name, these receptor proteins are the molecules that help the cell to receive signals from its surroundings. Often it is not necessary for the signal molecule to be let into the receiving cell. The message can be received as the signals knock at the outer cell boundary. Special receptor proteins lying embedded in the cell membrane do the job efficiently and the messages are passed on further into the cells. Setting up a well-tuned chain reaction, the surface receptor proteins transduce environmental signals into

32


chemical messages within the cell. The binding of the signal molecule to a receptor protein often changes its shape. This in turn triggers electrical or pressure changes in the micro milieu of the cell membrane. Sometimes the message is for the enzyme assemblies on the cell membrane itself; sometimes it is for the enzymes dispersed in the cytoplasm. Further, molecular communicators called 'second messengers' get involved and in turn pass on the message to other cellular proteins. The cell responds to these signals by modulating its chemistry appropriately. A number of proteins eventually convey information to the DNA molecules by acting as regulators. The signals from the surroundings influence the master molecule via the regulatory proteins that associate with it. More about this a little later! Proteins act as transporter of the cellular cargo. A large number of molecules come in and go out of the cells. Since the cells are busy metabolising, it is only natural that a large number of molecules should either be entering or leaving the cell at any given point of time. Interestingly, the rules of the passage in and out of the cell are rather strict. Only a few select molecules are permitted free trafficking across the membrane. Most others have to pass through the molecular gates. These gates are membrane proteins too! Further, the transport of molecules may be through 'channels' made of proteins that open and close only if correct signals are provided. At times energy too must be provided for proteins to shuttle ions. These proteins form the ionic pumps. Most of these transporter proteins are associated with other membrane proteins, which help in controlling the molecular movement. Proteins engage in a number of highly specialised functions. Some proteins, like haemoglobin in the red blood cells and myoglobin in the muscle cells, carry oxygen. Some proteins like myosine, actin, troponin and tropomyosine help muscles contract; immunoglobulins


33

help in body defence mechanisms. Some, like crystalline proteins form a transparent fluid in our eye lens. Our cells contain about a billion proteins—each important in its very own way. Once we accept and appreciate the bewilderingly diverse roles proteins play, the logical question that follows is: How do they do all this? How can a molecule made up of permutation and combination of merely 20 tiny chemical blocks manage to hold the cellular centre-stage? To function as they do, each protein must be made uniquely. For the protein to function well its amino acid sequence, its shape and size are important because most proteins do their work by interacting with other molecules. The molecule that associates with a protein is called a 'ligand'. All the work that molecules of proteins do is because of their binding to or release from their ligands. When a ligand binds a protein molecule it undergoes a change in shape. This modification can be used by the cells to get the protein to do all sorts of jobs. An enzyme can be activated by the binding of a ligand. Transferring a phosphate group from an energyrich molecule can activate proteins. A large family of enzymes is occupied in activating proteins by phosphorylating them and are known as 'protein kinases'. Some enzymes on the other hand, change the activity of proteins by removing the phosphate group. These enzymes are known as 'phosphatases'. Functions of a large number of proteins are governed by addition or deletion of phosphate groups from certain amino acids. Such a change brings about a change in their three-dimensional features. An alteration of this type is called an 'allosteric transition'. This change makes the protein ready for further reaction. For example, if a protein is to act as an enzyme, allosteric activation can increase its ability to bind the substrate. If the enzyme is to be stopped, a ligand binding can bring about change in its contour. The new allosteric change may reduce the affinity between

34


Empty effector s

Substrate binding site

Full effector site

Substrate site changes structure

Fig. 3.2: Allosteric proteins exist in alternative conformation.

the enzyme and the substrate. Allosteric changes can cause proteins to undergo definite movements (Fig.3.2). The allosteric changes in the protein myosin, for example, drives the contraction of the muscles. Ionic channel proteins undergo allosteric changes with the binding of a ligand. Certain protein conformations of the channel molecule allow the ions to pass while certain conformations do not. Ionic pumps too operate on the same theme. Protein communications are essentially ligand-induced allosteric changes. The entire plethora of protein action seems to be governed by just one ingenious method of molecular manipulation. But for it to work, it is necessary that the protein structure is accurately formed and shaped. Much of cellular machinery is devoted to regulation of proteins. It is one of the most critical processes taking place in the cell. As we come to know more and more about ourselves,


35

it is becoming increasingly evident that knowing about proteins, their synthesis and regulation is one of our most important achievements; perhaps the most important of all the puzzles we have solved. Well, perhaps 'solved' is too self-congratulatory, for there are missing bits to be fitted in the cellular jigsaw. Soon we probably would have a framework of how molecules regulate each other and express as physical reality in our cells. Scientists the world over have got together under the auspices of the Human Genome Project to work out all the codes in our DNA molecules. A new molecular anatomy is being unravelled; an exciting new puzzle is being solved—and that again is a story in itself, in which proteins have a leading role to play! A quick look at the specialised proteins helps us gain an understanding of the diverse roles proteins play.

The Defence Proteins

A large number of proteins participate in defending our body. One of the most important classes of defence molecules, without which we would die of infections, is the immumoglobulins (Ig). Known as the antibodies, they constitute about 20 per cent of plasma proteins by weight (Fig.3.3). Most abundant and active in fighting any foreign particle in the body (known as the 'antigen') is the simplest of immunoglobulins, the Y-shaped IgG molecule. It is made up of two 'light' and two 'heavy' polypeptide chains (socalled because of their molecular weights) held together by molecular bridges. Depending on the types and the number of chains in a molecule and their specific functions, four more types of immunoglobulins are seen in our body. They are the IgA, IgD, IgM and IgE. All immunoglobulins have a special site by which they bind the antigen. Molecules of IgG can bind microorganisms or foreign proteins and completely inactivate them by blocking all their important functional sites. This

36


Fab

Fab

Variable region Light chain

-

Constant region

Carbohydrate

Heavy chain

Fc Fig. 3.3: Structure of an immunoglobulin (IgG) molecule.

coating of the antigen by the antibodies helps the scavenger cells of the body, the macrophages, to identify and destroy them. IgG and IgM also have a site to which the other defence proteins of the body can bind. 'Complement' is a set of proteins that can bind the antigen-covered foreign cells or microbes and drill holes into their membrane, effectively killing them. All defence proteins work together to keep us free of infections.

The Respiration Molecule

Our lives depend totally on molecules that help us

PROTEINS AT WORK

37

Haem group with iron atom

Fig. 3.4: The tertiary structure of haemoglobin.

breathe. Of the consortium of molecules involved, an important one is haemoglobin. Packed in millions in the red blood cells (RBCs), these molecules shuttle the oxygen we breathe in and the carbon dioxide we breathe out (Fig. 3.4). Haemoglobin is made up the globular protein—globin—that snugly encloses the iron-containing haem. We can make five types of globin subunits, namely alpha, beta, gamma, delta and epsilon. Early embryonic and foetal cells make little of the beta chains but more of the other subtypes. In the adults 98 per cent of the globin molecules are of two 'alpha' and two 'beta' chains. About 2 per cent consists of two alpha and two delta chains while less than one per cent is made of two beta and two gamma chains.

38


A number of normal variants of globin molecules are seen in the human populations. However, a few can cause severe anaemias. Each of the globin subunits is associated with one, 'haem', and can carry an atom of oxygen. When RBCs reach the lungs, one of the haem units binds the oxygen and triggers the process of grabbing three more oxygen atoms. The bound oxygen makes the haemoglobin molecule more compact and bright red in colour. The oxygen is carried to the tissues and released while the carbon dioxide is taken up and carried back to the lungs to be disposed of when we breathe out.

A Communicator Protein

Proteins that communicate, travelling long distances via the bloodstream, from an organ that produces them to other organs, are hormones. One such communicator is insulin. Produced by the pancreas, it controls the glucose metabolism (Fig.3.5). Insulin is a small protein made up of two polypeptide chains held together. The pancreatic cells synthesize pro insulin, a precursor of insulin, and store it until required. With a fall in sugar levels in the blood, pro insulin is cleaved to form the insulin molecule and let out into the blood. Insulin reduces blood glucose levels by modulating the metabolism of muscles, liver and adipose tissues. Cells that respond to insulin carry on their surface a receptor for it. The insulin receptor is again a protein. One end of the receptor partly sticks out of the cell surface and binds to the insulin molecule. The other end protrudes into the cell. This part of the molecule can act as an enzyme when the receptor is bound to insulin and activates other cellular proteins by phosphorylating them; A cascade of molecular events follow, resulting in increased glucose uptake by the cells and conversion to storage form.

Horse

Horse

Ox insulin

Whale and pig

Disulphide linkages

Fig. 3.5: Structure of insulin

Disulphide linkage.

/

P R O T E I N S AT W O R K 39

40


Insulin effectively holds the blood glucose to a nearly constant level, irrespective of the fluctuation in the dietary uptake and cellular consumption of glucose. An uncontrolled sugar level, or diabetic condition, is caused if insulin synthesis is inadequate.

An Evolutionary Clock Protein

Proteins with the same function in a wide number of

Fig. 3.6.a: The cytochrome C protein.


41

species have probably evolved from common ancestral molecules and are called 'homologous . Such proteins help molecular paleontologists to work out evolutionary relationships between animals. Cytochrome C is one such protein (Fig.3.6.a). Cytochrome C is a coloured iron-containing protein present in the mitochondria. It is a part of the molecular chain that transfers electrons during biological

Monkey Homo sapiens, J Mouse chimps / 4

Mammals

Kangaroo 9\

Rabbit \3 Horse, pig, sheep, c o w C > Dog, seal, 1

Ostrich

Hippopotamus

reptiles

E m u ^ V Chicken, turkey Pigeon - ^ j / ^ P e n g u i n ZyjO-tt" Turtle Duck^W_. ° Rattlesnake

ir

'2

Bony

Birds and

18

Dogfish

Carp

Cartilaginous fishes

fishes Tuna-

- Lamprey

Amphibians Bullfrog

Starfish Earthworm -

8

23

Fungi

Yeast

Moth, hawkmoth Honey bee

„ 16/

16

Candida,

Insect

Clw

^ly

Locust Wheat

Neurospora

Vseasame Rice/ /4

Humicola-

^33

Sunflower

Plants

Spinach Ginkgo

Fig. 3.6. b: M a i n branches of the evolutionary tree based o n cytochrome C clock.

42


oxidation to generate energy-rich molecules. With a molecular weight of 13,000 it is made of 100 amino acids. The amino acids of cytochrome C from a large number of organisms are almost identical in 27 positions as these amino acids are functionally important. It has been estimated that 29 out of every 30 amino acids that change in cytochrome C are not tolerated and are eliminated during evolution. Differences in amino acids help us in making a rough estimate of how long ago the animal groups diverged. It has been further estimated that in about 21 million years one acceptable change per 100 amino acids takes place in cytochrome C. This is called the 'unit evolution time' for cytochrome C. On the basis of this an evolutionary tree can be drawn, indicating the time of divergence of organisms (Figi 3.6.b).

Proteins for Contraction

The contraction of our muscles is carried out by a number of proteins. A major protein involved in skeletal muscle contraction is myosin II. It has two heavy chains, each made up of about 2,000 amino acids. It also contains four light chains, two with 190 amino acids and two with 170 amino acids. The molecule is a 150 nm structure with a rod-like tail and two globular heads (Fig.3.7). Associated with myosin is another protein—actin. Each actin molecule has 375 amino acids and an energy-rich molecule, adenosine triphosphate (ATP), associated with it. Actin molecules can link together to form a filament. The actin filament and myosin II form the typical overlapping molecular array in the muscle. The sliding of these two molecules past each other causes the muscle to contract. There are other proteins that aid muscle contraction. Troponin, a complex of three polypeptides, helps control

P R O T E I N S AT WORK

43

Sk Thin filament

Thick filament

Fig. 3.7: The thick and thin filaments of a muscle slide past each other during contraction.

44


calcium levels in the muscle. Tropomyosin, a rod-shaped molecule, binds the actin in resting muscle. Titin, the largest protein yet known, acts like spring to keep myosine in form while another large protein—nebulin—regulates the actin assembly. Desmin filaments hold the myofibres in place and the molecular assembly is anchored to the muscle membrane via an elongated muscle protein—dystrophin. This orchestra of proteins ensures that each molecular step in muscle contraction takes place precisely.

A Dual Function Protein

Acetyl choline receptor is a special protein—an ion channel and a receptor for the neurotransmitter, acetyl choline, rolled into one. Densely packed, at the neuromuscular junction about 20,000 receptors per jim are found. The acetyl choline receptor is made up of five subunits. Of these two are alpha chains, and one each are beta, gamma and delta chains. Embedded in the membrane as a ring of molecules, these chains form a pore filled with water. The alpha chains have a site to which the acetyl choline released by the nerve terminal can bind. The binding of acetyl choline to the receptor changes the conformation of the protein complex and opens the gate for the ions to flow across. The ionic channel has a lining of negatively-charged amino acids that ensures that only positively-charged ions, especially of diameter less than 0.65 yim (nanometre), pass through. Normally sodium, potassium and calcium ions can cross over with ease. However, in the muscle cell, the forces working on the ions are such that a net inflow of sodium ions takes place. Thousands of ions rush in almost at the rate of 30,000 ions/channel/milli seconds. This ionic change triggers the muscle contraction. The channel remains open for about a milli second and

PROTEINS AT WORK

45

flips back to the original state. The acetyl choline detaches from the receptor and is degraded. The receptor is free to perform its dual functions, all over again.

3

MAKING PROTEINS Poems are made on trees, this paper was once a tree. Trees themselves do make lovely poems, A thousand poems will not make a tree. . . .But only a tree can make a tree. — G.B. Chelleja Poetically or otherwise, the fact that all life forms arise out of pre-existing ones is well appreciated. We also know that life we see around us today is very different from what it was millions of years ago. And sometime in that unrecorded, remote past, molecules came together to make cells. How it all happened is an intelligent guess based on the chemistry we know today. Imagination lets us lumber through those times as we wonder how proteins became a part of all life-forms. Think of the primitive earth witb its hot and sultry atmosphere, with not a breath of oxygen. Some molecules must have dissolved in the water and come together spontaneously as lightning struck the planetary terrain. The so-called organic soup that was formed probably had the first of macro molecules, jostling for space and fighting for the raw material that made them (Fig. 4.1). Some molecules could be formed by breaking down others. In this soup must have been the monomers or subunits of all macro molecules, of proteins, of carbohydrates, of lipids and of nucleic acids. It must have been a tough job for the macro molecules to survive as large structures unless something helped

MAKING PROTEINS

Amino acids

47

Proteins

Fig. 4.1: Atoms joined together to form amino acids.

them escape from being broken down into subunits. Some molecules did manage to do so by an ingenious method. They developed a coat of protein. The macro molecules that managed to somehow assemble this protein coat were the nucleic acids. These were perhaps the earliest cells. With time, innovations on survival strategies resulted in the formation of the cells as we see them today. Although there is no such thing as a 'typical cell', almost all cells share some common features. Cells are selfcontained blobs of jelly-like substance, called the 'protoplasm' containing all that is required for survival. Each of the cells taken from organisms evolutionarily higher than bacteria has a distinct 'nucleus'. The nucleus is that part of the cell that contains DNA. The boundary of the cell is defined by a double membrane which acts as a barrier,

48

THE SECRETS O F PROTEINS

restricting the movement of chemicals in and out of the nucleus. From the primitive cells that carried DNA without the nuclear demarcation to cells with the DNA closeted in the nucleus was an evolutionary leap. The nucleic acids, restricted to the 'nucleus' seem to mastermind the elaborate cell machinery. The cells also seem to carry a lot of apparently useless nucleic acid. This extra nucleic acid with no manifest purpose, seems to hitch-hike its way through life. It might, of course, manage to become useful sometime in millions of years and add a new asset to the cell machinery. By introducing variation over a large span of time and sifting the variants through the sieve of natural selection, more adapted life forms have appeared on earth. Probably this is how life has changed on our planet. If this is so, the nucleic acids are very smart molecules indeed! Long, delicate molecules containing sugar, phosphates Nucleic acid building blocks

5 carbon sugars

Ribose (RS)

Deoxyribose (DS)

Phosphate g r o u p

Fig. 4.2: Ribose sugars, deoxyribose sugars and phosphate groups make up the backbone of the DNA molecule.

MAKING PROTEINS

49

and the bases are nucleic acids. Chemically, the sugar is a little different from the one we eat. It is a pentose, made up of five carbons with four of them forming a ring and the fifth one sticking out. To this fifth carbon is attached a molecule of phosphate (Fig.4.2). The phosphate bridges the third atom of the carbon of the next sugar molecule. This five-carbon-ringed sugar is 'ribose' and together with the phosphate constitutes the backbone of 'ribonucleic acid' or RNA. When an atom of oxygen is removed from the sugar molecule, it forms the deoxy ribose sugar that forms the backbone of the 'deoxy ribonucleic acids' or DNA. The bases that form an integral part of the nucleic acids are organic molecules that are rich in nitrogen. The nucleic acids have five bases: thymine (T), adenine (A), cytosine (C), guanine (G) and uracil (U). Of these thymine, cytosine and uracil are alike and are called the 'pyrimidines', while guanine and adenine are similar and are called the 'purines' (Fig.4.3). Each subunit of nucleic acid containing a molecule of sugar, a phosphate and a base is called a 'nucleotide'. In other words, both the nucleic acids are chains of nucleotides. The two nucleic acids do not differ from each other only in the sugar they carry but also in the bases they are made up of. The ribonucleic acids have adenine, cytosine, uracil and guanine. The deoxyribose nucleic acids have thymine instead of uracil, the other three bases being the same. These small differences make the two molecules very different from each other. The differences are even more pronounced in the roles they play during protein synthesis. An appropriate interplay between the two types of nucleic acids create proteins. What the DNA molecule looks like and how it works has been understood after almost a century of experimentation. Initiated into an enquiry by Gregor Mendel on 'particles' of heredity, in 1865, scientists painstakingly worked for years on the question. While bacterial and fruitfly genetics ascertained the role of DNA as the

50


Pyrimidine bases

Thymine^:

.

uracil (U)-

Cytosme © Purine bases

Guanine (G)_ Fig. 4.3: The base^ are an integral part of the DNA molecule.

heredity molecule, biochemists and physicists developed novel protocols and techniques to understand the physical features of the molecule. By the middle of the 20th century the DNA molecule yielded to insistent probing. The elusive genetic code was cracked. In 1953, James D. Watson and Francis Crick proposed the double helical model for DNA (Fig.4.4). This brilliant proposal that set the stage for molecular biologists to take over was supported by inputs

M A K I N G P R O T E I N S 65

Fig. 4.4: The DNA molecule.

51

52


Gene

DNA •

Transcription

Primary transcrip-

Post transcriptional processing

Nucleotides'

A

Mature mRNA

mRNA degradation Translation

Protein (inactive)

Amino acids

Post translational processing

Modified protein (active)

Protein degradation

Fig. 4.5: Six processes that control the production of \ protein.

STUDYING

PROTEINS

from several contemporary laboratories. The mid 20th century was an exciting time! Years of work had culminated in a new insight into the molecules of life. The trigger prompted several scientists to look at the cell machinery with a new eye. The rapidity with which molecular biologists dissected the cellular microcosm is indeed breathtaking. The process of protein designing and synthesis was elucidated. It turned out to be a beautifully coordinated process tuned to the needs of the cell. A little complicated for beginners in biology to comprehend, the process is worth looking into to appreciate its sheer efficiency. Like a tiny well-managed factory, the cell makes all its proteins on demand! Making proteins is not an easy job (Fig 4.5). To make the protein of the right shape and size, the amino acids have to be lined up accurately. Each amino acid must be in the correct order and a bond that stitches them together must be formed. The two nucleic acids, DNA and RNA, divide the work between themselves. DNA helps out in providing the correct sequence of amino acids and RNA forms an elaborate machinery to stitch them up. To provide the sequence of each protein, the DNA carries a chemical code. Very simplistically, the stretch of DNA that carries information of one protein is technically called a 'gene'. Synthesis of a protein involves molecular deciphering of the code. The RNA molecules are deft in doing so. The molecules of DNA that hold the information for protein synthesis are organised as helical structures. Two strands, each of the sugar phosphate with the bases are wound in a helical form and are held together with bonds between the hydrogen of the bases. Adenine of one strand forms two links with thymine of the other strand. Likewise, guanine of one strand holds on to cytosine of the other strand by three hydrogen bonds. Since pairing specifications are stringent, the base adenine is said to be complementary to thymine and guanine is complementary to cytosine. This is called base pairing. In brief, molecular

53

54


biologists represent them as A-T, C-G. This sort of pairing ensures that one strand of the DNA molecule has a complementary strand with which to pair. It also ensures that if the strands are to be replicated they can do so by using one strand of DNA as the template. Further, this inbuilt quality to pair with the complementary base is the foundation of making a copy of the messages carried by the DNA. Each human cell (except the reproductive or the germ cell) carries 46 molecules of DNA. The DNA molecules in our cells are complexed with some proteins and can be seen, sometimes very distinctly, as chromosomes. The germ cells carry half the number of chromosomes (23) as compared to normal body cells. This is called the 'haploid number' of chromosomes. When germ cells from two parents meet, the original number of chromosome is restored and the baby has the diploid (46) number of chromosome. Each one of us gets 23 chromosomes from each of our parents. Some chromosomes are short while others are long. Together they carry all the codes to make into a human being. Bearers of hereditary, they are also the molecules that make each of us different and unique! For new proteins to be synthesized, the proteins associated with the DNA molecule must make way for the message to be exposed. Other proteins are pressed into service at this point. A battery of proteins help in the unwinding of the region that carries the code. Another set of proteins, special enzymes that link up the subunits of RNA, are geared into action. Using DNA as a reference, an RNA with the complementary base sequence is transcribed. This is the temporary copy of the code. The copy of the code is just a single strand of RNA molecule. This molecule of RNA is called the 'messenger RNA'. Obviously, the RNA copy is not biochemically identical to the DNA code but it carries the same information. This is because when the messenger is formed, the rule of the pairing of the bases is strictly followed. The copy of the

STUDYING

Amino acid Glycine Alanine Valine Leucine isoleucine Serine Threonine Aspartic acid Aspargine Glutamic acid Glutamine Lysine Arginine Cysteine Methionine Phenylalanine Tyrosine Tryptophan Histidine Proline

PROTEINS

Codon triplets in m-R NA GGA GGG GGC GGU GCG GCA GCC GCU GUG GUA GUC GUU CUG CUA CUC CUU AUA AUC AUU AGG AGA AGU AGC ACA ACG ACC ACU GAC GAU AAC AAU GAA GAG CAA CAG AAA AAG AGG AGA UGC UGC AUG UUC UUU UAC UAU UGG CAC CAU CCA CCG CCC CCU

Fig. 4.6: Amino acid codon triplets deciphered.

message formed is in the form of bases complementary to the DNA master plate. The messenger RNA is transported to the cytoplasm where a pool of amino acids is readily available. This is where the message gets read and the codes are translated into a sequence of amino acids. To be read three at a glance is an elegant mode, for each amino acid, three bases is the code. The message to be decoded is carried as a sequence of bases on the DNA strand (Fig.4.6). Three bases together code for one amino acid and is called a 'codon'. For example, the three uracil, one after another (UUU), is the code for the amino acid, phenylalanine. If two uracils are followed by an adenine (UUA), the amino acid coded for, is isoleucine. At times,

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more than one triplet can code for an amino acid. A permutation of three of the four bases A, T, C and G can specify each of the 20 amino acids that make the body protein. A triplet (AUG) signals the beginning of the message. It specifies the amino acid, methionine, and is known as the 'start codon' since in almost all cases the start site for translation is determined by AUG in the mRNA. Of the 64 codons that the permutation of four bases can generate, three do not code for any amino acids. These are UAA, UGA and UGA. These codons are called 'nonsense' codons or 'stop' codons and signal the end of the message. These codons are like molecular traffic lights, telling the cellular machinery when to proceed and when to halt. The genetic code was assumed to be universal for a long time after it was first deciphered. Years later we learnt that the human mitochondrial DNA has a slightly different code. The triplet UAG (a 'stop' codon) codes for the amino acid, tryptophan, while AGG and AGA act as 'stop' codons. Since then, however, only few differences have come to light and so the code for nuclear genes is considered to be universal. The picking up of the message by the RNA from the DNA molecule is a complex process. It requires plenty of help from enzymes and other molecules to get the message transcribed from the DNA language to the RNA vocabulary. Some molecules open up the DNA. Others get the monomers that are going to form the mRNA ready The key enzyme is the polymerase. To be more specific, it is the DNA-dependent RNA polymerase that has the main job to do. It travels over the DNA strand and uses it as the template to form the messenger molecule. It ensures that every monomer inserted in the messenger has the base complementary to the one in the DNA. The stretch of DNA acts as a template and the newly-formed mRNA is the copy of the message, ready to travel to the cytoplasm. This process of making the messenger is technically called

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'transcription'. The strand of mRNA may have faithfully transcribed the DNA message, but at this stage it is not ready to carry out its work. It requires enzymatic processing before it is ready to get on with its job. Enzymatic editing of the mRNA is necessary, specially when genes have regions that are not a part of the code that is used to make the protein. Such non-coding regions are removed before the mRNA is ready for transport out into the nucleus. The stretch of DNA that ultimately expresses as a protein is called an 'exon' while the intervening sequences are known as the 'introns'. A gene may consist of several introns and exons but the messenger that is sent out for translation carries only the copy of the exons. There is more to mRNA processing than removing introns. A string of amino acids is added to the newlyformed mRNA at one of its ends. This tail of adenine (poly A) is added enzymatically. The poly A tail protects the nascent mRNA from degradation and also helps in transporting it out into the cytoplasm safely. Much later, the tail is removed and this in no way interferes with the reading of the codes for protein synthesis. The other end of the mRNA is provided with a 'cap'. The cap is tagged on by the enzyme, guanyl transferase, adding a molecule of guanine to the last nucleotide of the mRNA. The cap plays a role in starting off the protein synthesis. It also offers protection to the mRNA from degradation. This processing of mRNA is necessary as it provides the cell with a strategic point for modulating protein synthesis. Interestingly all the processing of mRNA is done systematically by yet another set of protein-RNA complexes in the nucleus. These are called the 'small ribonucleo proteins'; snRNPs, for short! Chopped to requisite length and modified suitably the mRNA emerges out of the nucleus and homes to the specific cellular structures called the 'ribosomes'. Out in the cytoplasm, often lined up like an assembly line of a factory, are small cellular structures or the ribosomes. Their

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polypeptide chain

Messenger RNA

Anticodon

Transfer RNA

s

Amino acid

Fig. 4.7: Translation of RNA to protein.

job is to provide the infrastructure for reading the message that specifies the sequences of the amino acids of the protein (Fig. 4.7). A pool of energised amino acids is ready for the line up too. However, the amino acids all by themselves are helpless for they have no way of reading the triplet code. Another molecule chips in. Again an RNA. It is a tiny structure acting like an adapter. Peculiarly twisted, this molecule is the transfer RNA (tRNA). It has a special region that carries a triplet of bases, the so-called 'anti-codon'. This triplet anti-codon can recognise a code for an amino acid on the messenger. The tRNA molecule has another site where the amino acid can attach. Cells have 20 types of tRNAs, each carrying an amino acid with an appropriate anti-codon. The amino acids ready themselves to line up by attaching themselves with appropriate tRNAs. With its corresponding amino acid tagged to it, the tRNA, now called the 'amino acyl tRNA', closes up to the ribosome and the mRNA it holds. Each ribosome is made up of two parts; each part is chemically a protein-RNA complex. Binding of the mRNA

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to one of the units brings the two units together and the messenger RNA slips into a groove between the two composite units. The ribosome has two more slots, both for tRNAs. One of them is the peptide-binding site or P site, the other is the amino acid-binding site or the A site. When the tRNA associates with a ribosome poised to read the message, it does so by pairing its anti-codons with the codons on the mRNA. Once initiated, the protein chain forms along with molecular shifts. Very like an assembly line, the ribosome, the mRNA and the tRNA undergo repeated cycles of a typical procedure. This process is called 'translation'. A large number of small protein molecules are involved in getting translation done well. The most critical step in forming proteins is joining the two amino acids together. The bond that holds them is the 'peptide bond' that is formed when -COOH group of one amino acid reacts with -NH3 group of the other one. This happens when two amino acids are close to each other. The two tRNAs that occupy the P and the A sites on the ribosome are close enough to link up the amino acids they carry. The amino acids link up readily with the enzyme, peptidyl transferase, helping out. As the bond is formed between the amino acids, the tRNA of the P site is free to move out. The tRNA now carrying the newly tagged peptide chain, shifts into the vacated P site while a new amino acid carrying tRNA with an appropriate anticodon slips into the A site. The peptide bond can be formed again. As the cycles go on, the message is read to the very end. The result is a fresh, brand new protein. Once the message is completely read, the ribosome machinery is dismantled. The mRNA as well as the components of ribosomes are free to start all over again and make more proteins. So brisk is the procedure that at times several ribosomes are at work on the same message. Once translated the messages too are dismantled by enzyme action. The protein is almost ready and may have a long way to go.

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Protein synthesis is a rapid process although the rate depends on the ambient temperature. Bacteria kept at 37°C add about 15 amino acids to a polypeptide chain every second. The red blood cells add about two amino acids to a chain at the same time. This bit of the protein jigsaw was difficult to work out. World over, several scientists worked on the problem and put in their individual pieces to present an overall picture. Different bits of information emerged from a variety of sources. Recognised as well as unrecognised efforts helped to build up the story. Of course, there is more to it.

5 E D I T I N G PROTEINS Small as a peanut, big as a giant, We're all the same size, when we turn off the ligh';. — Shel Silverstein We are also the same size if converted into simple molecules. Most of the sugars, the fats, the proteins and even the nucleic acids that make up life forms are so similar that the variety of designs and functions that we see in the living world seems amazing. Equally surprising is the ability of these molecules to organise into different types of cells in a human body. Starting from a single cell that divides to form many more, a large variety of cells emerge, differing in their shapes, sizes and their working. Each is a special type of cell. The cells that make the lungs are different from those that make the bone or the muscles. Yet, they are very much alike. Each carries the same code for making proteins, and makes the majority of proteins that are identical. However, some proteins in many cells are special and not made by other cells. For example, the red blood corpuscles make haemoglobin that helps them carry oxygen. The muscle makes the protein— myosine—that helps them contract. The red blood cells do not need to contract and so, do not produce myoglobin. Likewise, the muscle cells do not synthesize haemoglobin. However, this does not mean that muscle cells lack the DNA that codes for haemoglobin. Like all other cells in the body, the muscle and RBCs too start off with a complete set of genes. RBCs, after they have synthesized the required

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quantity of haemoglobin, get rid of all their genes when their nucleus disappears to make more space so as to enhance their oxygen-carrying capacity. On the other hand, muscle cells carry copies of all the genes, even those not required by the muscle cell, for example, the haemoglobin gene, in an inactive mode. Quite literally, the genes that are not required are switched off. Sometimes some cells need certain proteins to be made intermittently. The genes needed are then switched 'on' or 'off' as and when required. 'Waste not, want not' appears to be the cellular motto. Gene regulation or the selective use of the DNA code to synthesize protein, is the core of all cellular and biochemical changes that take place. These small differences make a big difference to cell identity and the way the cell functions. For these cells to function normally, the right proteins must be synthesized at the right time and in the right quantity. How this is done is a question that is difficult to answer. Scientists have been studying the problem on different cells, using different methods. Conclusions of their experiments have been put together. What emerges is a hazy picture of how protein synthesis is manipulated in a cell to make it what it is. One of the major conceptual breakthroughs in understanding gene regulation came when Francois Jacob and Jacques Monod proposed the so-called 'operon model' for gene organisation and regulation. They shared a Nobel Prize in 1965 with their colleague Andra Lwoff for their work. How they pieced together the operon model is an interesting story. The system they were investigating were the common bacteria, E. coli. They observed a very curious thing about these little organisms. Normally, when provided with glucose in the medium, the bacteria used them as a source of food. Instead, if lactose, another sugar, was provided, the bacteria modulated their genes to synthesize a battery of enzymes that helped in lactose metabolism. On the other hand, if the bacteria were given a choice and

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supplied glucose as well as lactose in the medium, they preferred glucose and stopped synthesizing the enzyme required for lactose digestion. To put it differently, here were simple bacteria, with a single strand of circular DNA, responding to their environment by altering their gene expression. The operon model, which now appears an elementary molecular concept of gene regulation, was not easy to deduce (Fig.5.1). A large number of bacterial mutants with disrupted lactose metabolism had to be studied before a coherent picture emerged. We now know that three enzymes, all involved in lactose metabolism—B galactosidase, galactosidase permease and thio galactoside transactylase—are encoded by a contiguous stretch of DNA. These genes, called the lac Z, lac Y and lac C, respectively are linked and controlled as a single unit. The control was exerted by stretches of DNA adjacent to them. One such site was the 'operator' while the other was the 'promoter'. Regulatory gene

Control sites

;p

0

Structural genes

*

Lactose operon

v

a

5 TGTGTGlGAATTGT(^G0GjQj.Ajl|AACAATTTCACACA 3'

3

ACACAC CTTAACACTf QCCp AfTTGTTAAAGTGTGT 5' Protected by repressors

Fig. 5.1.a: Map of the lac operon with nucleotide sequence of the lac operator.

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i

y

z

P

a

Repressor binds to the operator and prevents transcription of lac z, lac y and lac a

imRNA

Repressor

/

P

o

y

z

imRNA

a

lac mRNA

^

^ P-galactosidase

Permease

^

Transacetylase

Inducer - repressor complex Fig. 5.1.b: The lac operon repressed (top) and induced

(bottom).

The lactose operon can be said to have three structural genes and the regulatory genes: the operator and the promoter. In generalised terms, operon is a set of genes under one single controlling regulatory region. Several operons have been studied, most of them in the E. coli, to understand how exactly the control on genes is expressed. The operator and promoter do not code for any protein at all. They are regulatory regions of the DNA. Operator is the region where a 'regulator' molecule would bind, while promoter is the region where the enzyme—polymerase—

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attaches at the start of the transcription. In the operon that controls lactose metabolism, the regulator molecule is a protein that sits on the operator and does not allow the transcription to take place when glucose is available and enzymes needed for lactose metabolism are not required. Such regulators are called 'repressors'. When lactose is available it enters the cells, binds to the repressor molecule and distorts it in such a way that it cannot bind the operator any longer. It de-represses the lactose operon and the enzymes required can now be transcribed. Though there is more to the operon of lactose, this scheme of controlling genes seems to be a common feature of bacteria. Several variants of this model have been discovered as bacteria were studied for their gene regulation. There are some regulators that help in transcription and are called 'inducers'. These themes with little modification seem to operate in our cells as well. Gene control in our cells is more complex because a lot more regulation is required. Unlike the bacterial cell, our cells have to handle a large number of signal molecules. Messages from the hormones or other similar secretions and from the surrounding cells have to be integrated into the cell system before a specialised protein is synthesized. The study of operons in bacteria has helped us enormously to understand the complexity of our genes and protein synthesis. How is protein synthesis or gene expression controlled in our cells? Well, in a complicated process, it is a good idea to be well organised. And when several things have to be managed simultaneously, it is better to get others to share the efforts. That is how the master molecule, DNA, seems to work. Not only is it organised but it also ropes in many other molecules to help it control the making of proteins. Transcription, the formation of the messenger RNA, we know, is the first step of gene expression which eventually culminates in protein synthesis. Decision of making

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or not making a protein, is taken in most cases, at this stage. The codes carried by the DNA are really not freely available. The DNA molecule has to be coaxed into revealing its secrets. Molecules, known as 'regulators', are good at this job. A large number of regulators are proteins. Some are RNA molecules. The rapport between the DNA and the regulators results in a well-timed protein synthesis. People we work or live with are easy to recognise. Meeting them face to face, or seeing them in profile or for that matter, getting a mere glimpse of their noses is enough to say who it is. The DNA molecule can also be recognised with ease by all the molecules that work and live with it. A mere brush of it in profile is sufficient for the regulators to recognise the site where they must interact with the DNA molecule. The messages carried by the DNA are recognised face to face when the mRNA is formed, while bases are recognised in profile by some regulatory proteins. Once recognised, these molecules loosely bind to the DNA. Just as in bacteria, regulators in our cells are really 'liaison molecules'. The protein requirements of the cell are communicated to the DNA by regulators which bind to the operator. Very like the bacteria, all our genes have a promoter and an operator. Some genes share their promoters and operators and can make messengers, all at the same time. The promoter is the DNA stretch in continuity with the gene; the operator may or may not be close to-the gene. But it does manage to come close enough to change the messenger formation. To start transcribing the message, the polymerase enzyme must bind to the promoter. It requires a signal from the regulators to do this. Some regulators bind the operator region and help the polymerase get started. Others stop the polymerase by blocking its binding to DNA. Once the signal of the regulator is sorted out, the polymerase can bind the DNA. However, a number of regulators do not act simply by blocking or deblocking the polymerase site. The

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exact mechanism of how several repressors act is yet to be elucidated. The mRNA formation cannot start without the help of several other molecules other than the polymerase. To get it started, a set of proteins called the 'transcriptional factors' help out. All these molecular communications ensure that the right message is formulated. In our cells binding of transcriptional factors acts as regulatory points. Often regulators increase protein synthesis by boosting the assembly of these transcriptional factors. Our genes have another feature that is far more complex than the bacterial genes. Our genes are many more in number and distributed not on a single strand as in the bacteria but are disposed in 23 pairs of DNA strands. Spaced as they are, our genes still control each other. A long-distance control can be exerted by some genes. A stretch of DNA, thousands of nucleotide sequence away, can influence the transcription of another region. Such long-distance controllers are called the 'enhancers'. A regulatory molecule binding to an enhancer can alter transcription of a gene very much away from it. Our genome appears to be a concoction of genes we know as 'structural' as well as a variety of 'gene control regions' often referred to as the 'regulatory genes'. Operators, promoters, enhancers and many such sites on the DNA help modulate protein synthesis and are regulatory in function. Regulatory genes have turned out to be most eiffective regions in the DNA for they have far-reaching effects on gene expression. A slight alteration in a structural gene affects only the protein it codes for, while a defect in a regulator gene affects all proteins under its control. At times, some genes have to be held in silence for a long time. For example, the gene that codes for haemoglobin must not express itself in any cell other than the red blood corpuscles. The way the DNA molecule is organised helps in switching some genes off almost permanently.

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Proteins called 'histones' are closely in touch with the DNA. Stretches of DNA coil around a few molecules of histones to form a structure that looks like a bead in three dimension. Each of the beaded structure of the uncoiled chromosome is called the 'nucleosome'. Separating two beads is like a small bit of DNA not tangled to proteins. The long molecule of DNA is a chain of beads that further coil and super coil. All this intimacy with the proteins gives the DNA molecule its typical profile. Sometimes the DNA coiling is so difficult to unwind that it becomes impossible to get the molecule to transcribe. Such a tightly-coiled DNA can be seen under the microscope as distinct from the less coiled part. Genes trapped in this part of the molecule cannot make proteins easily. Proteins that are necessary for a limited period of time are located in such a region. Once enough protein is synthesized, supercoiling of DNA molecule silences their codes. Supercoiling also silences genes not needed in the tissue at all. As mentioned earlier, genes are made up of exons (the protein coding sequences) and introns (the intervening, non-coding sequences). A gene may have any number of introns. The gene for human beta globin is 1,500 baseslong and has two introns. The message that is decoded to form the protein has only 600 nucleotides. The gene for albumin is made up of 25,000 nucleotides of which only 2,100 actually make the mRNA that is read by the ribosome. It has 14 introns. Every time the nascent mRNA is formed all the sequences are picked up from the DNA template, so are all the exons and all the introns. The messenger is then cut and trimmed to the right length. This modification of the message is one point where the size of the protein can be altered. In other words, gene expression can be modulated during mRNA processing. The same stretch of code can be used to make different proteins by cutting the messengers at different points.

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Manipulating life of mRNA is another form of controlling protein synthesis. Once formed, the messenger RNA starts its journey out of the nucleus. And at times it is not all that simple to get out. A large number of messages are just not permitted to go out. They are trapped in the nucleus and may be chopped into bits. Of course, they are treated very differently if the cell needs the protein to be made. The messages are then protected by providing them with protective 'molecular caps' and let out into the cytoplasm. The ribosomes then read and re-read the message. If the protein is required in a large quantity, as haemoglobin is, the messages have a long life. Otherwise they are dismantled soon after they are read. Finding out all about how making of proteins is controlled in a cell has been tricky. Some information has been gathered by working on simple cells like the bacteria, some has been collected by working on yeast. Work on the fruit fly, Drosophila, has also been very rewarding. Some studies have been carried out on cells in culture: normal as well as abnormal cells have been studied in detail. And the story that has emerged is not easy to put together. What happens in the cells can only be deduced by analysing all aspects of molecular interactions. In the tiny world of a cell what really happens is at best a confused molecular bombardment that results in a well-regulated formation of the brand new protein. The job, however, is not done until the proteins start carrying out their functions.

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C O M M U T I N G TO W O R K If you were only one inch tall, you'd ride a worm to school. The tear drop of a crying ant would be your swimming pool. — Shel Silverstein To be made of a particular size is important or the world becomes a different place. This is true for all of us. More so for a protein molecule. Not only is its size important but so is the shape. If the specified shape and size are not conformed to, the protein molecule is in trouble. It can barely perform its function. At times it even fails to reach safely to the place where it has to work. To get a protein of the right size, shape and function not only must be its code transcribed properly from the DNA, it must be translated well too. A lot more happens to the protein molecule soon after, it is made and this ensures that it reaches parts of the cells where it is needed. Proteins are, in fact, needed everywhere in the cell. Some are needed to form part of the cell organelles. For example, mitochondria, the powerhouse of the cells requires proteins as a part of its membrane as well as in the spaces within. Ribosomes, of course, require proteins. Proteins are needed to form all membranes within the cells and are also part of the plasma membrane that forms the outer boundary of the cells. They participate as a part of the golgi complex to form granular material that gets pushed out of the cells. Some proteins are required in the

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cytoplasm itself. Sending proteins to the place of their work is a job by itself. And knowing about how this is done has been of recent interest. A key role in protein transport is played by the 'endoplasmic reticulum'. The word 'reticulum' means network. Endoplasmic reticulum is the network of tubules within the cell that helps in protein transport. It is a structure made up of membranes with a lumen within. Like any other membrane in the cell, the membranes of endoplasmic reticulum are made up of lipids with proteins embedded in them. Most proteins that are to become a part of the membranes of the cell are carried by endoplasmic reticulum. So are the lipids. Proteins that are to be transported out of the cells are also sent via this network to the golgi complex. The golgi complex is a set of tubules that form vesicles of material to be taken out of cells. The golgi complex and the endoplasmic reticulum work hand in hand to export proteins. The endoplasmic reticulum is so keenly involved in protein trafficking that at times it becomes a part of the protein-making machinery. It gets drawn into the process of translation soon after the first few amino acids are linked. In fact, this newly synthesized stretch of protein acts like a signal that ushers the ribosomes to associate with the membrane of the endoplasmic reticulum. All proteins destined to be transported by the endoplasmic reticulum carry this signal peptide. With further help from other molecules, called 'docking proteins,' the ribosome, well into the process of making the string of amino acids, launches itself onto the membrane of the endoplasmic reticulum. The newly formed protein has no chance of drifting away, for close to where the ribosome sits on the membrane is a channel that leads into the lumen of the endoplasmic reticulum. Once the protein is well inside, the signal sequence is removed and the protein released. It is now free to move and is carried further to its destination. The endoplasmic reticulum not only transports protein but it is here that it gets linked to other molecules that

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make it ready for its function. Proteins in the cell are not always present as such but form liaisons with other molecules. Some get associated with lipids while others link up with carbohydrates. Molecules that bring about correct associations are abundant in the endoplasmic reticulum. As newly formed proteins enter the lumen, these molecules recognise sites where molecular ties have to be made. As the appropriate molecules link up with it, the protein folds to get into its correct shape and is now ready for work. Several proteins become a part of the structures within the cell. The nucleus, where the DNA resides requires proteins. Some proteins are required to perform duties related to the upkeep of the DNA molecule. Some are required to become a part of the nuclear membrane and control flow of substances in and out of the nucleus. Proteins on the job in the nucleus must reach their location correctly. Most of these proteins are synthesized in the cytoplasm. They all carry the 'nuclear localisation signal'. These signals are not present as a single stretch as in the case of proteins that enter the endoplasmic reticulum. Instead they are present as specific groups of amino acids on the protein chain when the protein is being formed. When the newly formed chain of amino acids folds and becomes three dimensional these special stretches of amino acids come close together to become the sorting signal. Nuclear localisation signals are not removed after the proteins have reached the nucleus. This may be so because the nucleus disassembles and reforms every time the cell divides. The proteins need to be guided back to their places after each such upheaval. The cell gets all the energy for its business from small power stations dispersed in the cytoplasm. These are the mitochondria. Almost like tiny independent organisms inside the cells, the mitochondria have their DNA and protein synthetic machinery. They can transcribe some of the proteins they need using their own DNA. But they also need some proteins to be transcribed by the DNA in the nucleus. As they perform a complex function of making

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energised molecules, the mitochondria need lots of special proteins: some to be working right inside in the matrix, some lodged in the 'inner membrane' that carry the power-making assembly of molecules, some work in the spaces between the membrane. Proteins made in the cytoplasm need to reach the right place in the mitochondrial compartments. A newly formed protein to be imported into the mitochondria has a stretch of amino acids at one end of the molecule that acts as a signal. On the outer membrane of the mitochondria are proteins that can bind these signal molecules and lure them into the mitochondria. Proteins homing to mitochondria do not fold into their final conformation until they reach their destinations. Helping them remain in the unfolded state and move inside through the membranes are other proteins, called the 'chaperone' proteins (Fig.6.1). Once attached to the outer membrane, the new protein passes in through the two mitochondrial membranes in one go. Once inside, the signal peptide is removed and the protein folds. It is now in place and ready to function. In case, a new protein has to be inserted into the inner membrane, either its transport to the matrix is halted or the protein that has reached the matrix is regained. Another stretch of amino acids closely following the one that helps homing to the mitochondrial membrane helps out in this. Sorting of proteins in the mitochondria is a complex process carried out with remarkable efficiency by a number of molecules. Interestingly, most of the molecules helping in transport are themselves proteins. Another organelle performing a crucial function in the cell is the peroxisome. It essentially breaks down fats. Just a bagful of chemicals that help in this process, peroxysomes have a single outer membrane. Proteins for peroxisomes are made in the cytoplasm. Just like the other proteins that are transported into endoplasmic reticulum, these proteins too have a small sequence of amino acids that acts as sorting signal. As all proteins that travel to different

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No chaperone

Chaperone binds

Protein acquires mature structure: chaperone dissociates

Fig. 6.1: Chaperone binds to ensure correct folding of the protein. Once the molecule is folded, the chaperone dissociates.

organelles have their special 'ticket' in the form of special signal peptides, the ones that don't have remain in the cytoplasm itself. In other words, if proteins have their working place as the cytoplasm they do not carry any special sorting signal or molecular tag. After they are formed they just fold and move with the surrounding fluids. The cell, producing thousands of proteins in a lifetime, several of them made simultaneously, really has a tough job

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i

to do. It manages to do so remarkably well! The cells do slip sometimes. Not all proteins are correctly made. Errors may creep in as the message is copied from the DNA or as it is getting translated into the sequence of amino acids. The result is poorly made, inefficient molecules. Some are unable to assemble well and fail to work. Some misfold. Chaperone molecules in the cytoplasm try to salvage these proteins by helping them to attempt refolding. If they fail, the malformed molecules have to be broken down in the cell. Moreover, all proteins have a life span. After a time when their role in the cell is over, they have to be dismantled. Some proteins have a very short life and are present transiently in the cells. Proteins that must be got rid of are marked out by molecular tags. One such tag that attaches to most incompetent proteins is a very small protein called 'ubiquitin'. This protein can bind to some amino acids that are exposed when the protein is misfolded. Tagging of ubiquitin to any protein attracts the executor molecules of the cell. Protein complexes or groups of proteins acting together, called 'proteasomes', patrol the cytoplasm for ubiquitin signals. When proteasomes encounter a ubiquitin-tagged protein, they start chewing it up and break it into small fragments. This system is so efficient that newly formed chains of amino acids have to be guarded by the ribosome machinery or the chaperone molecules lest they get tagged by ubiquitin. Some proteins get protected because they have amino acid sequences on their ends that resist degradation. Such sequences are carried by long-lived proteins as well. Bombarding each other, meeting at crossroads, the molecules of life are always active. Their world is so small that forces that may by chemical standards be called weak, influence their activity very strongly. A change in the pattern of folding, an extra charge here or there, or a wee bit of error makes a big difference to what happens to these tiny molecules. The outcome of these interactions is what makes a cell vibrantly alive. And it is a variety of proteins that are the heart of all this activity in the cell.

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STUDYING PROTEINS Lives of great men all remind us We can make our lives sublime, And on departing leave behind us Footprints on the sands of time. — Longfellow A number of scientists, both men and women, have left behind their footprints in a more tangible way. As publications of their works, sometimes remarkable, sometimes not so noteworthy! Although some scientists engaged in a lifetime of research are never recognised formally, for they may have missed out on dramatic breakthroughs, most of them have contributed to science in their own small way. It is often on the basis of this collective contribution that ideas that make a difference emerge. Technical developments, at times small and apparently insignificant, provide insights into ticklish problems. A shift in paradigm is often preceded by emergence of a new technique. Study of proteins has been possible because of the variety of techniques developed to study them. What we know of proteins today has been based on years of work in developing appropriate systems to tackle protein problems. How do we know so much about proteins? How do we know their chemical compositions, their chemical shapes or their molecular secrets as they go about their cellular duties? There is no doubt that kudos go to scientists who over the years refined their techniques and honed their skills, such that almost no aspect of protein structure or

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function lies beyond their ken. However, this does not mean that we know everything there is to know about proteins. Many details are shrouded in mystery. The complete answer still evades us. But progress is being made. Slowly and steadily. Newer and better technologies rub shoulders, so to say, in the laboratories. The result? A clearer picture and better understanding. The first question that surfaces in the common man's mind is: Can I see protein? Then answer will follow instinctively: Oh! How can it be? The reality is that the question is not as naive or as preposterous as it may appear at a first glance. Scientists routinely see proteins. They can see it localised within the cells or see them after they have been extracted out of the cell. Cells are about five times smaller than what we can possibly see with the naked eye. Molecules of proteins, much smaller, obviously cannot be seen unaided. An important science, microscopy, has developed over the years to see the enlarged and resolved images of the material we cannot see with the naked eye. Starting from a simple microscope fabricated by Leeuwenhoek in the 17th century, an astounding variety of microscopic techniques have developed, each more refined and giving a better picture of the cellular micro structure. In most of the microscopic techniques for protein visualisation the fundamental principle is to somehow selectively link the protein molecule with a molecular tag. The tag may be a dye, such as coomassie blue, that stains most proteins to enable proteins in the cell to be seen coloured blue under the light microscope. The dye used could be the one that emits fluorescence, such as fluorescein. Proteins stained with the dye can be seen as fluorescent material using a fluorescence microscope. A similar underlying principle is used in autoradiography—a technique where radioactive molecules are used to tag proteins. By using isotopes of different elements it is possible to tag different proteins. This is possible because

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some proteins are modified and contain small groups that change the ionic charge of an amino acid. For example, the amino acids—serine, tyrosine and occasionally threonine— are phosphorylated. That is, a phosphate group is added, usually to their respective hydroxyl group. Such a protein is said to be a phosphoprotein. Using radioactive phosphorus to tag protein would thus mean that phosphoproteins are selectively tagged. Autoradiography uses isotopes to tag proteins and then traces their presence on a photographic film. Regions of radioactivity produce silver grains in the film and these can be observed as dots representing areas where the tagged proteins are present in the cell. An elegant modification of the microscopic technique is 'flow cytometry'. It is a rapid method that can an be used to get a comparative profile of the amount of protein in each cell. Cells stained by a protein-specific fluorescent dye are made to flow through a chamber where the fluorescence emitted is collected, amplified and converted into an electronic pulse. The pulses, each corresponding to the amount of protein specific dye in the cell can be further analysed by the help of a computer. Flow cytometry techniques have turned out to be very important especially when clinically important proteins have to be studied rapidly for making diagnosis of various diseases. One of the most dramatic techniques that helps localise proteins into precise cell compartments is electron microscopy Three- and two-dimensional structures of protein assemblies on the cell membrane or as cytoskeleton can be visualised using electron microscopy. Cells prepared for electron microscopy are stained with electron-dense material and subjected to a beam of electrons. The stained structures scatter part of the electron beam while the rest is used to form an image. The electrons are then picked up on a photographic film where the stained regions show up dark as compared to the rest of the background. All these techniques are used routinely now. The protocols of staining specific protein have been modified to

STUDYING PROTEINS

79

ensure best results. Newer dyes and staining methods are available. Staining of specific proteins is now possible by using what is known as immunohistochemical methods. These methods exploit the fact that antibodies against a protein bind exclusively to the molecule. Antibodies carrying a tag can be used to localise the protein accurately in the cell. Immunohistochemical methods are most widely used to identify proteins today. Study of proteins i within cells provide clues to their functions. The story is incomplete unless the structure too is known. Working out the sequences of amino acids in a protein and the understanding how it folds in three dimension has occupied several protein chemists. They have a tough job to do. Firstly, from all the mess of molecules in the cell, the protein of their interest has to be separated and purified. It is then characterised for its physical features, like molecular weight and size. Finally, the molecule has to be broken to study composition and sequence of the amino acids. Today scientists also believe that the story is not complete until the gene coding the protein is identified! This is indeed a tall order! Although a number of methods are used to do all this, some have proved to be more favoured by scientists and are routinely used. The first step in cell-free protein study is to disrupt the cells and get the protein into solution. This is rather simple as cells are fragile and will break open easily. Cells can be just macerated. They can be shocked into breaking open by exposing them to ultrasonic vibrations or solutions of low ionic concentrations. Forcing them through a small orifice or repeatedly freeze-thawing them can also do the trick. Once the macro molecules are out in solution the cell debris can be removed by centrifuging the homogenate. Now the more difficult step starts. Getting the purified protein. In the last two decades, techniques of protein separation have become greatly simplified and refined We know a lot about protein behaviour in solution and t'lis helps us

80


to isolate a particular protein and then study it. The most common method of separating out proteins is 'column chromatography'. It was invented in 1906 by I. A. Tswett and has now undergone a number of modifications. The principle underlying all the column chromatography techniques is simple. The mixture of proteins is allowed to percolate through chemically inert material slowly. Each type of protein in the mixture will react differently to physical features of the column and will be accordingly retarded in its movement through it. The protein with minimal retardation will flow out of the column first and can be collected as a fraction. Slowly and steadily all the proteins of the mixture can be eluted out, each as a separated fraction. Column chromatography has turned out to be a versatile technique. It can be modified according to known properties of the protein one is interested in. A variety of inert material or matrix can be used (Fig. 7.1.a). If a porous matrix is used, as in gel chromatography, the smaller molecules of protein are retarded in the column as they get trapped in the pores of the gel. This type of gel can be used to separate proteins on the basis of size. Another modification called 'ion exchange chromatography' is used to separate proteins on the basis of their charges. The matrix used carries charge and the proteins are retarded according to the charges they carry. One of the most elegant modifications of column chromatography is 'affinity chromatography'. The matrix in this case carries a specific antibody or any other ligand to which the protein to be separated can bind exclusively (Fig. 7.1.b). The protein that reacts with these ligands will be held back in the column as the protein mixture passes through. This can be later removed to get a fairly pure fraction. A single run on affinity column can effectively separate the protein of interest. Proteins can be separated on the basis of other properties too. In solution form most proteins will be charged

STUDYING PROTEIN?

Protein mixture is added to column containing cross-linked polymer. Protein molecules separate by size; larger molecules pass more freely, appearing in the earlier fractions. 1

2

3

4

5

6

Fig. 7.1.a: Gel filtration separates proteins according to size.

dl

82


Mixture of proteins

Solution of Iigand

Key.

Protein of interest

Ligand

Ligand coupled to polymer bead

Protein mixture is added to column containing a polymer-bound iigand specific for protein of interest. Unwanted proteins are washed through column.

Protein of interest is eluted by Iigand solution.

Fig. 7.1.b: Affinity chromatography separates proteins by their binding specificities.

STUDYING PROTEINS

83

molecules. If they are subjected to an electric field the protein molecules would move to the positive or negative pole depending on the net charge they carry, their size and their shape. This fact was exploited for the first time, in 1933, by A.P. Tiselius to develop the technique of electrophoresis. Later sophistication in the matrix in which this separation is carried out improved the procedure several folds. Today the most commonly used method is technically called 'SDS polyacrylamide gel electrophoresis'. The charge proteins carry depends not only on their structure but also on the hydrogen ion concentration. A measure of hydrogen ion concentration in a solution is known as 'pH' of the solution. It is possible to modulate the pH in such a way that the prdteins in solution have no net charge. Such a pH is called an 'isoelectric pH' of the protein. Proteins as well as amino acids have a typical isoelectric pH. As they carry no net charge at this pH, proteins at their isoelectric pH do not migrate in an electric field. This behaviour of proteins is used to separate out different proteins by the method known as 'isoelectric focusing'. The protein mixture is allowed to move through an electric field in a gel that has a pH gradient. The proteins simply stop moving in the electric field when they reach their individual isoelectric pH. This is a very efficient method of extracting pure protein. Once the protein has been acquired in fairly pure form, the next step is to find its weight, shape, size and other features like viscosity. A very simple way of characterising the molecular weights of proteins is by ultracentrifugation. Centrifugation is a scientific jargon for spinning anything. In an ultracentrifuge, the macro molecules are spun extremely fast. So much so, that centrifugal fields exceeding 250,000 times the force of gravity can be attained. At such high centrifugal fields protein molecules sediment out from solution. High centrifugal forces oppose the force of diffusion which keeps the proteins evenly dispersed in a solution. Further, to help separation the procedure may be

84


Sample

Direction of migration

Fig. 7.2: In electrophoresis, different samples are loaded in depressions at the top of the gel before an electric field is applied.

carried out in a medium where the density is low at the top of the test-tube (in which the protein is loaded) and goes on increasing at the bottom of the tube, i.e. it shows a gradient. When sedimented under these constraints, the rate of

STUDYING PROTEINS

85

sedimentation of proteins is a function of both their size and shape and is expressed as the 'sedimentation coefficient'. Proteins have sedimentation coefficients that range between 1 and 200 x 10-13 seconds. Sedimentation coefficient is higher for proteins with higher molecular weights but not proportionally so. Scientists use the Svedberg equation to find out the molecular weight of protein from its sedimentation coefficient. Developed by Theodor Svedberg in 1926, this technique is still in use and gives a good measure of the molecular weight of proteins. At times ultracentrifugation methods are used to make partially pure protein preparations that can be further purified by chromatography. Scientists are interested in knowing the viscosity of proteins for it gives an insight into their molecular properties and behaviour. Viscometers, instruments that measure viscosities, determine the relative rate of flow of a protein solution as compared to a standard. The viscosity of a protein solution is the best clue to its asymmetry and its change in shape. Compact protein molecules (enzymes, for example) have a low viscosity. Altered viscosity can give a clue to interaction between proteins. Solutions of muscle proteins, actin and myosin, are far less viscous when measured alone. Their interaction to form actinomyosin increases the viscosity of the muscle protein solution. Study of such reactions by viscosity measurement have been used to work out factors that affect protein interactions. Once the protein has been purified to a certain degree it is possible to get it to form crystals. Studying crystals takes scientists deep into the protein structure, to the realm of atomic arrangement of the molecule. X-rays are radiations with wavelengths much smaller than visible light. When a crystal is bombarded with X-rays most of them would pass through. Some however, will be deflected because of the atoms they bang against. The diffracted rays can be picked up on a photographic plate and the

86


pattern of diffraction studied. This pattern provides clue to the arrangement of atoms in the protein molecule. Another method to probe deep into protein structure is infrared spectroscopy. This technique is based on the observation that vibration of chemical bonds in a molecule can be seen as absorption when visualised in infrared light. Proteins have characteristic absorption bands depending on the direction of their peptide bonds. Infrared spectroscopy gives a clue to how the peptide bond in a protein is placed and this in turn, reveals how the protein molecule is folded. Small proteins or protein domains can be studied by using another spectroscopic method. Nuclear magnetic resonance or NMR spectroscopy measures the physical properties of the hydrogen nuclei in a magnetic field. Signals from hydrogen in different amino acids can be analysed to deduce distances between them and to eventually work out the conformation of the protein. The ultimate understanding of a protein requires the content and sequence of its constituent amino acids. To gain the sequence, the protein can be first digested by a mild enzyme, like trypsin, that breaks it into much smaller peptides. These bits of proteins can now be separated using chromatographic or electrophoretic techniques. Finally the fragments can be analysed in automated 'amino acid sequencers'. The sequencer is based on an elaborate procedure. To put it simply, an amino acid on one end of the peptide is tagged with a dye, the bond that holds the amino acid is then broken and the tagged amino acid is identified using chromatography. This cycle is repeated over and over again till the end of the amino acid string for a particular peptide. Then the information is pieced together to get the exact sequence. The procedure is long and at one time was done manually. However, it is now done far more rapidly and precisely using automation. As one can guess, amino acid sequencing is a laborious procedure. Even with automation the time taken is

STUDYING PROTEINS

87

significant. An alternative strategy has been developed with the advent of molecular techniques of DNA sequencing. Relatively easy, DNA sequencing can not only give the amino acid sequence of the protein but also the gene that codes for the protein. And this is ultimate in knowing about any protein! The trick used to get to the gene is quite logical. After a part of the amino acid sequence of a protein is known, its DNA code can be deduced. A labelled piece of DNA with complementary code can then be prepared and used to localise the DNA in the genome that codes for the protein. DNA sequencing of this region can now be done to get the amino acid sequence. This strategy works because the procedure for sequencing DNA is far simpler than sequencing proteins! Proteins are now being studied using computers. The central problem in protein chemistry today is designing artificial peptides that fold appropriately. Provided with information of chemical and physical constraints, protein folding can be predicted using computer modelling. How Proteins can be Studied • Proteins can be separated on the basis of their ability to dissolve in various solvents. This technique is called chromatography. Several types of chromatographic methods have been evolved to get highly purified proteins. • Most proteins are negatively charged and move in an electric field at rates that depend on the net charges they carry and on their size. They can be separated on this basis by the technique of gel electrophoresis. This technique is used routinely for protein separation. • Amino acid composition of a protein can be studied by first cutting the proteins into their subunits by using enzymes called proteases and then separating them by chromatographic techiques. The sequences of amino acids have been worked out by biochemical analyses or by automated amino acid analysers.

102 T H E S E C R E T S O F P R O T E I N S

Shapes of proteins can be studied by the technique of X-ray crystallography or by nuclear magnetic resonance spectroscopy. Proteins in a cell can be seen by staining them with a dye that binds only the protein molecules. Proteins have been visualised in cells using high resolution electron microscopy. How proteins behave in a cell can be worked out by tagging them with radioactive molecules and tracing them by a technique called autoradiography.

8 PAINFUL PROTEINS At the point of dying let death itself perish with its last torments Only then will life come to take its place. — Rabindranath Tagore Eternally human beings have fought death and disease. Surviving involves fighting for good health. With civilisation we have developed conscious health care systems. Some of them have been formally recognised as science, others not. All the same, freedom from ailments has been on our minds for long. One aspect of health we have miserably failed in tackling up till now is inherited diseases—most of them arising out of defective protein synthesis. As we know more and more about proteins and the genes that code them, it is becoming apparent that a number of our ailments including cancer and mental disorders are also because of defective proteins. The cells of our body synthesize a large number of proteins. The process of protein synthesis involves a number of molecules that carry out the function quite efficiently. But like any other system, the strictly regulated protein synthesis too is prone to errors. Abnormal proteins may be formed because the process of transcription or translation at times slips. When errors in protein synthesis occur in somatic cells the problem is not always very serious, though at times it may result in set of diseases, such as cancer. Abnormal protein synthesis in germ cells has a more far-reaching effect.

90


Most causes of abnormal protein synthesis can be traced to the molecule of DNA. The genes or the associated regulatory system could have undergone an alteration. The alterations in the base sequences of DNA are known as 'mutation'. These changes are fairly permanent and heritable. When mutation arises in germ cellsd (eggs or sperms), the baby inherits it. If mutations affect proteins that are functionally important the baby may be born with a disease. Today we know of a number of diseases, some of them debilitating, that arise because of defective protein synthesis. In 1902, Sir A. Garrod, recognised now as the father of human genetics, described the inherited disease, called 'alkaptonuria'. Patients suffer from progressive damage to joints and spine and discolourment of skin and eyes. Sir Garrod described it as a disease caused due to abnormal metabolism of protein. Much ahead of his time, he further suggested the presence of a strong linkage between the metabolic deficiency and its inheritance. We know today the details of the metabolic defect. The enzyme called homogentisic acid oxidase, essential for the breakdown of amino acids, tyrosine and phenylalanine, is absent in those affected. The accumulation of homogentisic acid, an intermediate of the metabolic pathway is responsible for the symptoms. A large number of diseases, all caused due to absence of an important protein in a metabolic pathway, are now known. Another special protein in our body and one which is prone to abnormal formations is haemoglobin. The spectrum of anaemias resulting from its malfunction are classified as haemoglobinopath. Normal adults have the molecule of haemoglobin made up of four chains of the protein globin, two of them called the 'alpha chains' and two, the 'beta chains'. A change of even a single amino acid in the globin molecule can affect the oxygen-carrying capacity of haemoglobin.

PAINFUL PROTEINS

91

Replacement of the amino acid valine with glutamine in the sixth position in the C globin chain produces an abnormal molecule called HBS. Its presence alters the shape of RBC. As the affected RBCs look sickle shaped, the disease is called sickle-cell anaemia. It was the first disease where molecular abnormality in protein was traced back to a point mutation, a change in a single base of the globin gene. Another haemoglobinopathy prevalent in the Indian subcontinent is thalassaemia. The main symptom of the disease is anaemia, ranging from mild in some, to lethal in others. The cause is abnormal synthesis of the globin chains. When the alpha chain is reduced or missing, the person is said to be suffering from alpha thalassaemia. The gene coding for the alpha chains of the globin molecule is located on the 16th chromosome. The absence of a gene or a mutation that results in abnormal protein being formed reduces the amount of alpha chains in the RBC. The beta chains may be produced normally but the imbalance caused between the two chains is not tolerated by the body and becomes the cause of the disease. A similar situation arises when the beta chain is missing or abnormal. It causes beta thalassaemia. The beta gene is located on the 11th chromosome and can malfunction because of a variety of possible mutations. The mutations may disrupt the translation, or may produce a distorted code or change in the gene such that the mRNA processing is abnormal. Another set of diseases, known as muscular dystrophies, is caused because another special protein which is absent or is produced in low quantity. The skeletal muscles of those affected waste away. Manifestation of the problem ranges from mild weakness of the muscles to a near crippling condition, resulting in death early in life. The severe form is known as Duchenne muscular dystrophy (DMD) while the milder form is known as Becker muscular dystrophy (BMD). The muscle protein dystrophin is

92


absent in DMD and is present in low quantities in BMD. A large protein, 3685 amino-acid long, dystrophin, is located in the membrane of the muscle cell. Its main function is to mechanically stabilise the muscle membrane specially after contraction. A muscle lacking dystrophin is sensitive to contraction and may be easily damaged and can waste away. The gene for dystrophin is located on the X chromosome, which makes it a sex-linked disease, predominantly striking the males. Women are unaffected but may be carriers. The protein production in the affected cells is sabotaged as the gene has lost some of its parts. Such a 'deletion' may arise spontaneously and is then transmitted through generations. Just as proteins involved in mechanical work are important, so are the gatekeeper proteins. Channels, pumps, and transport molecules in our cells must be normal for good health. Otherwise a disease as severe as cystic fibrosis can develop. Cystic fibrosis is a disease caused due to abnormal epithelial cell function. Epithelial cells are cells that form internal and external linings of most of our organs. Apart from other problems those affected by cystic fibrosis have severe lung disorder. There is an over production of sticky mucus in the lungs and patients with a severe form of the disease do not survive. At the molecular level, the cause of abnormal epithelial cell function has been identified as the absence of a protein involved in chloride ion transport. It is a large protein consisting of 1480 amino acids and is called the 'cystic fibrosis transmembrane conductance regulator'. The gene that codes for it is located on the 7th chromosome. Several mutations that bring biosynthesis of the protein to halt have been identified. The defence mechanisms in our body are protein based. An abnormal protein synthesis in cells involved in immune function can cripple the system and leave us totally unprotected against infections. A rare genetic disease is

PAINFUL PROTEINS

93

'agammaglobulinaema'. The patients suffering from the problem lack the protein, agammaglobulinemia tyrosine kinase. As a consequence the cells that should produce immunoglobulins fail to do so and the patient is left susceptible to repeated viral and bacterial infections. The gene for the protein has been traced to the X chromosome. A far more crippling disease is 'severe combined immunodeficiency disease' (SCID). The disease may be lethal unless special care is given. The patient has to be transferred to a sterile chamber soon after birth. The affected do not produce 'adenosine deaminase', a enzyme important for nucleic acid metabolism. Absence of this protein prevents the immune cells from dividing normally and the patient fails to put up a fight even against a mild infection. The gene that synthesizes adenosine deaminase has been found to be on chromosome number 20. Abnormal protein formation has been implicated in the most complex areas of human disease, i.e. in psychiatric disorders. Neurons of the human brain are in touch with each other via the messenger molecules and membrane signals. It is not surprising that most of the cognitive functions require neurons to be engaged in special protein synthesis some time or the other. That abnormal protein synthesis can lead to dementia has been amply demonstrated by study of Alzheimer's disease. Alzheimer's disease normally strikes the old but in some families is known to have an early onset. In the affected, neural degeneration sets in followed by decline in all mental functions.The brain of the affected have 'senile plaques' and the neurons are packed with 'neurofibrillary tangles". These structures are essentially formed out of a protein called the 13 amyloid protein. A small pleated protein made up of 39 to 43 amino acids, it is derived from a much larger 13 amyloid precursor protein. The precursor protein is coded by the gene on chromosome 21. Mutation of this gene causes overproduction and deposition of fi amyloid and is believed to be the cause of

94


familial Alzheimer's disease. Evidence is also accumulating to show that complex mental illnesses like schizophrenia and manic depression may also have roots in unusual protein synthesis. Proteins that act as regulators and second messengers in our cells are most important for our well being. At times the changes in protein synthesis in the somatic cells have drastic effects. One such group of diseases, collectively called cancer, arises out of abnormal protein synthesis in somatic cells. Molecular biologists studying cancer have been screening for factors that lead to 'transformation' or conversion of a normal cell to cancerous cell. Environmental factors, viruses, radiations, chemicals, genetic predisposition—all have a role to play in transforming a cell. However, if the issue is probed deeper one common cause is uncovered. All these factors essentially cause alteration in protein synthesis. A host of proteins, with diverse functions, majority of them transcription factors, enzymes, and growth factors are produced unnaturally in the cancer cell. The genes that code for these proteins are within our cells and are called 'oncogenes'. When not producing the protein in an unregulated manner they are called 'proto-oncogenes'. Activation of oncogenes by any factor leads to unrestrained protein synthesis by these genes, and this in turn leads to cell transformation. The transformed cells now go berserk and do not follow any norms of normal division and growth. All cells derived from the one transformed cell behave in the same manner. They inherit the morphological and behavioural madness of the first transformed somatic cell. While certain genes have been seen to be familial, most cancers are caused by alteration in protein synthesis of somatic cells. Somatic-cell protein synthesis is extremely critical in another instance. The early stages of embryonic development are extremely sensitive. Any disruption in the synthesis of protein at these stages can lead to serious

PAINFUL PROTEINS

95

consequences. It is now known that all divisions and the formation of correct tissue at the appropriate time in embryonic life are because of precise protein synthesis. Inaccurate protein synthesis during development can surface as congenital defect. This is merely a glimpse into the spectrum of problems caused because of some protein or the other in our body not being normal. What can we do about it? What use is knowing so much about the defective protein, the gene that synthesizes it and the mutations that cause it if we cannot take steps to rectify the situation? It appears we do have some solutions though we will have to work much more for others. In most of the cases, an attempt is made to reduce the severity of the symptoms. For example, correct diagnosis of metabolic diseases early in life, often is very helpful. Sometimes, a special diet may give significant relief. In cases of haemoglobinopathies, repeated transfusions seem to be the only answer. Until recently, in most cases of inherited diseases there was little that could be done. As our knowledge about proteins, their synthesis and regulation grows, it introduces another form of therapy for diseases, and this is called 'gene based therapy'. The possibility of using or manipulating our genes for therapeutic purposes has been one of the rationales to undertake the daunting task of mapping all our genes under the auspices of the Human Genome Project. A revolution in medicine is probably in the making. However, the promise is not easy to fulfill, especially keeping in mind the complex way our genes are regulated to synthesize a specific protein at a specific time. Yet there is a lot of hope and we have had some success! The strategy of gene-based therapies is simple. The defective cell is provided with copies of the normal gene which synthesizes the protein. This in practice is not easy. The normal gene needs to be somehow pushed into the cell. It should be in the right place on the chromosome and should produce protein in required quantities at the right

96


time. This is known as 'augmentation therapy'. One of the first clinical trials of gene therapy was carried out on four-year old Ashanti DeSilva suffering from SCID, by W. French Anderson and his team at the Gene Therapy Laboratories at University of South California School of Medicine (USA). White blood cells of Ashanti were taken, copies of the gene required for the synthesis of the enzyme, adenosine diaminase, were introduced into the cells and the altered cells were injected back into her body. The therapy seemed to work well although she would need regular booster doses. It raised hopes of more possibilities. Most diseases with a single gene defect can potentially be taken care of by this approach. While gene therapy may have a long way to go before it fulfills its promise, there are other ways in which genetic information can prove to be of immediate relevance. There is no doubt that not only the patient but also the families of those affected face a traumatic situation. Genetic counselling and prenatal diagnosis would be of immense help, especially to people with a family history of an inherited disease or to couples belonging to communities with high possibilities of carrying the defective gene. Correct and reliable prenatal diagnosis can help take a decision and avoid births that would bring sorrow and pain. This would go a long way to solve the problem. Until recently, prenatal diagnosis was plagued with doubts about detection of mutations, but with more precise molecular techniques available, detection is now almost foolproof. Screening populations and spreading awareness through communication and education are also likely to help ease the situation. The painful proteins we inherit will have to be tackled on alj possible fronts!

8

PROTEINS—FOOD FOR THOUGHT 'For oh', say the children, 'we are weary And we can not run or leap If we cared for any meadow, it were merely To drop down in them and sleep.' — Elizabeth Browning This poem describing the miserable childhood of children forced to work for a livelihood is said to have awakened the world and altered the fate of generations of children. A hungry childhood is equally devastating and the world has yet to provide food for all its children. For a number of reasons, some universal and some local, millions over the world sleep hungry. Dietary habits of populations in different regions of the world have been determined mainly by the availability of foods locally and due to local practices. A great deal of trial-and-error experimentation must have shaped man's habitual dietary patterns. Mere satisfaction of hunger is not a safe guide for the selection of proper food. Sound nutritional principles alone can help us achieve a healthy and active life. The first attempt to recommend dietary allowances of protein, minerals and vitamins for Indians was made by the Nutrition Advisory Committee of the Indian Research Association (now ICMR) in 1944, following the recommendations of the League of Nations in 1937. Alongside, a typical balanced diet based on traditional Indian diet was also formulated to provide all the nutrients to meet the Recommended Dietary Allowances (RDA) of

98


an Indian male between 20 to 39 years of age and weighing 60 kg. Healthy, physically fit, he is employed for eight hours at moderately active work. He spends eight hours in bed, four to six hours sitting/moving about and two hours in active recreation or household duties. Since then, the protein energy and other nutritional needs of the Indian man have been periodically re-evaluated. In judging the adequacy of dietary proteins to meet the nutritional demands of man, not only the quantity but also the quality of the dietary protein(s) matter. All foods except refined sugar, oil and fats contain proteins in varying degrees. Foods such as meat, fish, eggs, pulses, nuts and oilseeds are protein rich. So is milk, if allowance is made for the large amount of water in it. Cereals and millets contain about 10 per cent protein. Rice has about 7 per cent protein which is less than that in wheat but its quality is better. The quality of dietary protein depends on the essential amino acids it supplies. The best quality protein is one that provides an essential amino-acid pattern very close to the pattern of the tissue proteins. Human-milk protein scores very highly in this regard. So it is used as a reference protein for defining the quality of other proteins. The minimum amount of essential amino acids required by infants are also taken as a reference pattern for defining the quality of proteins. The quality of dietary proteins are computed on the basis of the extent to which its essential amino acid pattern deviates from that found in breast milk or egg. This mode of assessment does not take into account the digestibility of protein which is an important criterion. Of course, the digestibility of certain proteins improves on cooking. However, excessive exposure to dry heat may make the lysine and methionine in vegetables unavailable to the body. Proteins are required for maintenance of tissues in adults and for growth in children. The dietary requirement of proteins increases in lactating females. Thus the relative requirements of a growing child, an adult male and a

99

PROTEINS—FOOD FOR THOUGHT

lactating female will be different. The actual amount of protein to be consumed daily to meet the requirements will also be governed by the quality of protein consumed. The higher the quality of protein in the diet, the lower the requirement and vice versa. The average daily protein requirement of an Indian adult, in terms of high quality protein like milk or egg is 0.5 to 0.7 gm/kgs. Table 1 Protein Requirements of Adults Body weight (kg)

Group

Requirement

(gm)

per kg

per day

Men

60

1.0

60

Women

50

1.0

50

Table 2 Protein Requirements during Pregnancy and Lactation Daily additional

Group

requirement Pregnant women

(gm)

Total daily requirement

15

65

0-6 months

25

75

6-12 months

18

68

(gm)

Lactating women:

Table 3 Protein Requirements (gm/kg) of Infants Age

(months)

Daily

requirements

0-3

2.3

3-6

1.85

6-9

1.65

9-12

1.50

100


Table 4 Protein Requirements of Boys Age

Body weight

Requirement

(yrs)

(kg)

(gm/kg)

Total

requirement (gms)

1-2

10.54

1.81

19.1

2-3

12.51

1.67

20.9 23.8

3-4

14.78

1.61

4-6

19.20

1.52

29.2

7-9

27.00

1.48

40.0

10-12

35.54

1.46

51.9

13-15

47.88

1.40

67.0

16-18

57.28

1.31

75.1

Table 5 Protein Requirements of G i r l s Age (yrs)

Body weight

Requirement

Total

requirement

(kg)

(gm/lcg)

(gms) 18.1

1-2

9.98

1.81

2-3

11.67

1.67

19.5

3-4

13.79

1.61

22.2

4-6

18.69

1.52

28.4

7-9

26.75

1.48

39.6

10-12

37.91

1.45

55.0

13-15

46.66

1.33

62.1

16-18

49.92

1.21

60.4

It is self evident that we cannot formulate a nutritionally balanced diet if we do not know the nutritive value of the individual items on the menu. Figures for protein requirements are valid only when other nutrients in the diet are adequate. It has been found that a mixed vegetarian diet can meet the protein requirements of adults and older children if the protein intake contributes 10 per cent


101

Lysine Methionine

B

B

A

A Adult requirement for lysine and methionine

Protein source A

Protein source B

Proteins A and B (mutual supplementation)

Fig. 9.1: Mutual supplementation of low-quality proteins is essential for a balanced diet.

of the total calories. For growing children and pregnant/ lactating women, proteins of animal origin are a desirable part of the diet. In all, a judicious mix of cereals, pulses, vegetables, with milk, egg, meat and fish makes up a balanced diet. Vegetarian diets that include milk or milk and eggs can supply the required amount of essential amino acids without difficulty. For strict vegetarians careful planning is required so that complementary protein sources are included at each meal. For example, legumes, low in methionine but adequate in lysine may be teamed with cereals which are low in lysine but adequate in methionine (Fig. 9.1). The biological value of proteins of some foods can be realised from the figures given below: Total protein 100 Egg 95 Cow's milk 84 Fish 76

102


Meat Rice Wheat Maize Groundnut Pulses

74 68 66 60 55 49-63 Table 6

Protein Composition of Various Fruits (per 100 gm of edible portion) Fruit

Proteins (gm)

Amla

0.5

(Emblica

officinalis)

Apple

0.2

(Malus

sylvestris)

Apricot

1.0

(Prunus

armeniaca)

Banana (Musa

1.2 paradisiaca)

Dates (dried) (Phoenix

2.5

dactylifera)

Grapes (Vitis

0.6-0.9

vinifera)

Guava

0.9

(Psidium

guajava)

Mango

0.6

(Mangifera

indica)

Mausambi (Citrus sinensis) Orange (Citrus

> 0.7

aurantium)

Papaya (Carica

0.8

0.6 papaya)

PROTEINS—FOOD FOR THOUGHT Table 7 Protein Composition of Various Vegetables (per 100 gm of edible portion) Vegetables

Proteins (gm)

Ash gourd (Benincasa hispida) Beetroot (Beta vulgaris) Bitter gourd (Momordica charantia) Bottle gourd (Lagenaria vulgaris) Brinjal

0.4 1.7 1.6 0.2 1.4

(Solanutn melongena) Cabbage (Brassica oleracea)

1.8

Table 8 Protein Composition of Various Oilseeds (per 100 gm of edible portion) Oilseeds

Proteins (gm)

Coconut (dry) (Cocos nucifera) Cottonseed (Gossypium) Groundnut (Arachis hypogaea) Mustard (Brassica nigra) Sesame seeds (Sesamum indicum) Sunflower seeds (Helianthus annus)

6.8 17.5 25.3 20.0 18.3 19.8

103

104


Table 9 Protein Composition of Various Pulses (per 100 gm of edible portion) Proteins

Pulses

(gm)

•

17.1

Bengal gram (Cicer arietinum) Black gram (Phaseolus mungo) Field bean (Dolichos lablab) Green gram (Phaseolus aureus) Horse gram (Dolichos biflorus) Kidney bean (Phaseolus vulgaris) Lentil (Lens esculenta)

24.0 24.9 24.0 22.0 22.1 25.1

Table 10 Protein Composition of M i l k (per cent) Source

Proteins (per cent)

Buffalo Cow Goat Human

4.78 3.47 4.03 2.14

These tables give us an idea about the protein content of the items included in our daily diet. Contrary to earlier beliefs that the adult brain is insensitive to even the most severe starvation, there are indications that it is vulnerable at a certain period of growth. Extrapolating data gathered from experiments on rats, this period falls between mid-


105

pregnancy and the second birthday. So protein/calorie needs must be adequate if peak health and performance are to be achieved. Malnutrition is one of the major problems that confronts us, more so in the relatively poorer countries. However, malnutrition strikes not only those who do not have enough but also those who have a diet deprived of proteins. This is a special type of malnutrition called the 'protein energy malnutrition' or PEM. Protein demands of the body not being met especially in childhood result in the deadly disease called 'kwashiorkor'. Curing this disease has been high on the agenda of policy makers fighting food problems. That a deficiency of proteins in diet can cause an almost incurable disease, killing young children, came as a surprise to people working in the field. It took time for it to be accepted as a problem distinct from just starvation. Cicely Williams, working in Ghana, published an article in The Archives of Diseases of Childhood. She reported a typical condition of children aged one to four, fed exclusively on a mildly fermented mash of maize. These children had 'edema', swelling due to fluid accumulation, specially on hands and feet. They had severe diarrhoea, sores and a peculiar peeling of skin. The disease was fatal. Survivors seemed to respond to a diet of codliver oil and canned condensed milk. These observations, that for the first time described a major protein deficiency disease, went essentially unnoticed, except that H. S. Stannus, an authority on tropical deficiency diseases, criticised it as being a case of infantile pellagra, a well-known condition. C. Williams, however, persisted in her study. She published her observations on 60 patients, and called the disease 'kwashiorkor'. In the local language the word meant 'the disease the older child gets when the next child is born' or 'deposed child'. Yet unrecognised, this protein problem received little attention till years later. Hugh C. Trowell, working under a similar

106


situation, believed the disease to be a complex syndrome and called it 'malignant malnutrition'. It was distinguished from pellagra—a disease due to deficiency of the vitamin nicotinic acid. As Trowell reviewed literature, he not only rediscovered kwashiorkor but also realised that the disease had been described by several workers. It was rampant and the main killer of children almost all over the world. Kwashiorkor became the focus of people fighting malnutrition. Why should low protein diet have such a devastating effect? To understand this it is worthwhile taking a look at. what happens to all the proteins that we eat (Fig. 9.2). Proteins are digested in our stomach and in our small intestine. To begin with, proteins lose their complexity of shape, i.e. they denature because of acid in the gastric juices. The gastric juice also has 'pepsin', an enzyme that attacks bonds between the amino acids. The protein chains become shorter after gastric digestion and pass on to the intestine. In the intestine the proteins are bathed in the juices from the pancreas. The enzymes—'trypsin' and 'chymotrypsin'—in the pancreatic juice further act on the peptides and chop them into still smaller fragments. The few dipeptides that escape this degradation are broken down by the enzyme dipeptidase to individual amino acids. Almost all the proteins from animal sources such as eggs, fish or meat are degraded to amino acids. Fibrous proteins like keratin are not completely digested. Some proteins from seeds or grains, being enclosed in cellulose, escape digestion. The amino acids formed are transported efficiently across the cells of the small intestine. They either diffuse in slowly or are picked up by molecular transporters. Structurally similar, amino acids are carried in by the same transport route. This would mean that excess of one of the amino acids will compete for the transport molecules and deprive a similar one of its entry into the bloodstream. In other words, deficiency of an amino acid


107

Salivary glands

Protein digestion is begun in the stomach by hydrochloric acid and the enzyme pepsin

The liver regulates the distribution of amino acids to the rest of the body .Stomach

ffttiver

Protein-digesting enzymes are secreted from the pancres into the small intestine

"Pancreas Absorbed amino acids enter the portal blood a n d t r a v e l t o t h e liver

The small intestine is the major site of protein digestion

Final digestion of dipeptides and tripeptides to amino acids occurs inside the mucosal cells of the intestine Little dietary protein is lost in the faeces Fig. 9.2: An overview of protein digestion and absorption.

108


may be experienced because of an imbalance in amino acid content in the diet. In kwashiorkor, the problem is slightly different. The gastro-intestinal tract is damaged because of lack of proteins and the normal absorption of proteins becomes impossible. A vicious circle starts where the child has diarrhoea and does not recover even if protein is supplied. Only a light protein diet is of some help. The amino acids entering through the intestinal wall, via the bloodstream reach various tissues and enter the cellular amino acid pool (Fig. 9.3). The cells draw upon this pool of molecules to synthesize new proteins. Proteins in the cell are periodically degraded and add to this pool of amino acids. It has been estimated that of about 300 gms of proteins made by the body, 200 gms are made from amino acids that are recycled within the body, while about 100 gms are made from amino acids that are supplied by the diet. If the diet does not provide the amino acids there Body proteins Protein synthesis

Dietary orotein Digestion

Protem degradation

AMINO ACID POOL

Synthesis of molecules such as neurotransmitters

Deamination Carbon compounds

Glucose synthesis

Energy production

Nitrogen

Fat

syndesis

Urea synthesis in liver

Urea excretion in kidney

Fig. 9.3: A m i n o acid pool in the body is replenished by amino acids from the diet and from the breakdown of body proteins.


109

I Intake

Nitrogen (mg/kg)

I Excretion

Positive nitrogen balance (growth)

Nitrogen equilibrium (maintenance and repair of tissue)

Negative nitrogen balance (wasting of tissue and loss of weight)

Fig. 9.4: Diagrammatic representation of nitrogen, balance.

is stress on the body to make up for the deficit. The 20 amino acids that we utilise can be said to be structurally carbon compounds containing nitrogen. Removing nitrogen from the amino acids or 'deamination' leaves behind a carbon skeleton that can be converted via metabolic processes into fats or carbohydrates. It is not surprising then that an abnormal protein diet affects not only protein metabolism but also affects the metabolism of other macro molecules. The nitrogen removed during deamination of amino acids is used up by the liver. The process generates a waste product called 'urea'. This molecule leaches into the blood and is eventually thrown out of the body through urine

110


and faecal matter. The majority of nitrogen lost by the body is in the form of urea. This observation has formed the basis for calculation of nitrogen balance of the body (Fig. 9.4). The nitrogen balance or the estimation of nitrogen intake as amino acids and output through urine and faeces is considered to be an indicator of protein requirements of the body. The status of protein metabolism of an individual and his/her protein requirement can be approximated by finding out the nitrogen balance. Let us consider an individual consuming 50 gms of protein per day. Proteins normally contain 16 per cent of nitrogen. This would mean that nitrogen intake of the person under consideration is 50 X 0.16 or 0.8 gm of nitrogen per day. If this person excretes an equivalent amount of nitrogen he can be said to be consuming just enough proteins. If the nitrogen lost is less than what is taken in, the person is said to have a positive nitrogen balance. Individuals recovering from infections or injury, pregnant women and lactating mothers require a positive nitrogen balance as protein utilisation is substantial. On the other hand, if the amount of nitrogen lost from the body exceeds the amount that is ingested, a negative nitrogen balance is said to exist. A person with negative nitrogen balance is likely to lose weight. To estimate the protein requirements of an individual a nitrogen balance study on the person must be conducted. As it is practically impossible to do so for everybody, a detailed study of a sample population is done and recommendations are made on the basis of this survey. In general, adults require 0.8 gm of protein per kg body weight per day. A person weighing 60 kgs would require about 48 gms of protein every day in the diet. Determining protein requirements based on nitrogen balance considerations alone has been challenged by some scientists. Not only do they feel that,the methods of nitrogen measurements are not foolproof but also feel that the balance of individual amino acids would be more


111

relevant. The alternative proposed means following the fate of each amino acid instead of calculating an overall nitrogen balance. This procedure involves injecting the person with a particular amount of amino acids labelled with a harmless radioactive carbon. The amount of labelled carbon expired as well as in repeated blood samples can be estimated. Metabolism of the individual amino acids can be worked out from this data. This sort of analysis revealed that the dietary requirement of the amino acids—leucine, lysine and threonine— is far more than was expected on the basis of nitrogen balance analysis. These conclusions, however, raised controversies, because the method of analysis was complex. As an excess of amino acids is as harmful as their deficit, protein dietary recommendations remain a concern. Why protein energy malnutrition gives rise to a diseased condition can now be appreciated. Prolonged and sustained inadequate diet not only changes the cellular biochemistry but puts the whole physiology out of gear. The body mechanisms that try to keep a balance, and maintain an homeostasis, are evoked. All sorts of metabolic adjustments are made so that survival is possible even under dire conditions. Starvation for the first 24 hours uses up the circulating glucose and fatty acids. Liver and muscle glycogen too are used up as fuel. Fats stored as triglycerides in tissues are pulled out into circulation as fatty acids and ketones. However, the brain selectively uses glucose for its function. As fats, unlike amino acids, cannot be converted into carbohydrates, the body proteins are forced to supply the amino acids that are further catabolised to glucose to meet the demand of glucose by the brain. Unlike fats there is no storage form of proteins in the body; as a result, a starving person loses weight. Over a period of time the body tries to make further adjustments. Some tissues, such as the heart, kidney, and muscles shift their fuel requirements from glucose to fatty acids and ketone bodies. Other tissues, such as the bone

112


marrow, switch to pathways that require very little oxygen. With extended starvation, the metabolic rate declines by 10 per cent. To help conserve body proteins, the brain eventually gives up using glucose as a fuel and switches to using keto acids. Body proteins are used more effectively, saving energy and reducing amino acid requirements. Nitrogen loss through urine too is reduced. All these changes are typical of the body trying to survive acute shortage of food. This leads to a typical pathology that is seen in malnutrition. Pathological changes that arise when food is inadequate lead to a wasting disease called 'marasmus'. On the other hand, specific protein deficit leads to the pathological condition as seen in kwashiorkor. In fact, a range of pathological conditions that arises out of lack of protein or energy are classified as 'protein calorie malnutrition' or protein energy malnutrition. The protein calorie malnutrition can be further graded, depending on the clinical manifestation. Most systems of the body are affected, specially if PEM is of prolonged duration. Most apparently affected is the gasto-intestinal tract. The 'villi' or finger-like projections in the intestine that are essential for absorption, become markedly blunt. The gastric juices and pancreatic secretions get decreased and contain less of digestive enzymes. As a result of these changes absorption of carbohydrates and vitamins too gets affected. The defence mechanism of a person with PEM is severely compromised. The major defence protein, immunoglobulin, is reduced and the body can hardly put up a fight against foreign organisms invading it. The levels of hormones that control various physiological processes show alterations. Impairment in reproductive potential is commonly seen in individuals with PEM. Function of the heart is affected as the heart muscles undergo atrophy and swelling. As the diaphragm and respiratory muscles are affected, respiration too is not normal. One of the most affected systems in PEM is the nervous


113

system. It is well established that right from the time the system is developing in the foetus it is sensitive to nutrition available. Rapid brain development takes place in the foetus starting from 30 weeks of gestation and continues till two years in the baby. PEM in mother in the first trimester of pregnancy adversely affects the brain growth of the foetus. Malnutrition that continues into childhood damages the cells of the brain. Though it is debatable whether these changes are reversible, there is no doubt that the these children have a tough time learning. Intellectual and cognitive development of these children is not normal. There may be other factors too that contribute to this situation. It has been also realised that damage is not restricted to the first two years, but affects behaviour and other brain functions, for the children are simply short of energy to learn. Proteins are indeed important for thought! Skills are less developed not only because of brain damage but also because the overall health is poor. A majority of these symptoms are reversible. A good balanced diet swings the system back to normal physiology, though some changes do stay on as permanent damages. Now that we know so much about proteins and their utilisation by the body, it is becoming easier to handle problems related to protein energy malnutrition. The next obvious challenge is to decide which foods provide adequate proteins. Several measures have been evolved to assess food quality with respect to proteins. One measure used is the chemical score. This is the ratio of an essential amino acid present in the body in least amount and the amount of this amino acid in the food under scrutiny. Protein efficiency ratio can also be calculated by comparing the weight gained by laboratory animals fed with a particular protein with the weight gain when fed with standard known protein. Some use a measure called 'biological value'. This is a ratio of nitrogen intake via a test protein and the nitrogen absorbed. Each of these

114


measures have their share of drawbacks but are useful to draw out some dietary plans. As none of the dietary conclusions concerning proteins are free of controversies, recommendations of what ideal diet should be has changed with times. It appears that for a normal person the time-tested traditional idea of moderation in all that we eat works out to be the best. Maintaining a variety in foods is seen to go a long way in taking care of this protein problem.

Further Reading 1. C. Branden and J. Tooze: Introduction to Protein Structure, Garland Publishing, New York, 1991. 2. T. E. Creighton: Proteins: Structures and Molecular Properties, second edition, W. H. Freeman & Co., New York, 1993. 3. J. Kendrew (ed.): The Encyclopaedia of Molecular Biology, Blackwell Scientific Publication, Oxford, 1994. 4. B. Alberts, D. Bray, J. Lewis, M. Raff, K. Roberts & J. Watson: Molecular Biology of the Cell, third edition, Garland Publishing Inc., New York & London, 1994. 5. B. S. Mahajan & M. S. Rajadhyaksha : New Biology and Genetic Diseases, Oxford University Press, 1999. 6. P. M. Udani (ed.): Textbooks of Paediatrics with Special Reference to Problems of Child Health in Developing Countries, Vol. I, Jaypee Brothers Medical Publishers, New Delhi, 1991.

Index Agammaglobulinaema 93 Allosteric 3 3 , 3 4 Alpha helix 20,24 Amino acids 5, 6, 7 , 1 2 , 1 3 , 1 5 , 1 6 , 1 8 , 1 9 , 20, 22, 24, 33, 42, 44, 53, 55, 57, 58, 59, 71, 72, 73, 74, 78, 79, 86, 87, 93, 98, 106, 108, 109, 111,112,113 Anti-codon 5 8 , 5 9 Autoradiography 77, 78, 88 Beta sheet 20, 24 Biological value 113 Cancer 94 Chaperone 73, 74, 75 Chromatography 1 2 , 1 6 , 8 0 , 8 5 , 8 6 , 87 Codon 55, 56 Collagen 22 Complement 36 Cystic fibrosis 92

Enhancers 67 Enzymes 2, 2 9 , 3 0 , 3 1 , 3 3 , 4 0 , 5 4 , 56, 58,59,63,65,66,85,87,90,93,94, 96,106,112 Essential amino acids 12,98,113 Evolutionary clocks 2 , 4 1 Exon 5 7 , 6 8 Flow cytometry 78 Gene 5 3 , 5 6 , 5 7 , 6 2 , 6 3 , 6 4 , 6 5 , 6 6 , 6 7 , 68,79,87,89,91,92,93,94,95,96 Haemoglobin 18, 32, 37, 38, 61, 62, 67,90 Haploid 54 Hormones 2, 3 0 , 3 1 , 38, 65 Immunoglobulins, 3 2 , 3 5 , 3 6 , 9 3 , 1 1 2 Infrared spectroscopy 86 Insulin 1 6 , 2 2 , 3 1 , 3 8 , 3 9 , 4 0 Isoelectric pH 83

Diet 1 , 2 , 5 , 9 7 , 9 8 , 9 9 , 1 0 0 , 1 0 1 , 1 0 4 , 105,106', 1 0 8 , 1 0 9 , 1 1 3 Diploid 54 DNA 2 5 , 3 2 , 3 5 , 4 7 , 4 8 , 4 9 , 5 0 , 5 3 , 5 4 , 55, 56, 57, 61, 63, 64, 65, 66, 67, 68,70, 7 2 , 7 5 , 8 7 , 9 0 Docking protein 71 Domain 20, 22, 24, 86

Kwashiprkor 1 0 5 , 1 0 6 , 1 0 8 , 1 1 2

Electrophoresis 83, 84, 86, 87

Nitrogen balance 109

Ligand 33, 3 5 , 8 0 Malnutrition 1,105,106, 111, 112,113 Marasmus 112 Muscular dystrophy 91 Mutation 9 0 , 9 1 , 93, 95

118


Nitrogen fixing bacteria 5 Nutrition 1 , 2 , 4 Oligomeric 22 Oncogenes 94 Operator 6 3 , 6 4 , 6 5 , 6 6 , 6 7 Operon 6 2 , 6 3 , 6 4 , 6 5 PEM105,112,113 Peptide bond 1 2 , 5 9 , 8 6 Peptides 1 3 , 1 6 , 2 0 , 7 1 , 8 6 , 1 0 6 pH 84 Polymerisation 22 Polypeptides 1 3 , 2 0 , 2 2 , 4 2 , 6 0 Primary structure 2 2 , 2 4 Promoter 6 3 , 6 4 , 6 5 , 6 6 , 6 7 Proto-oncogenes 94 Protomers 22 Quaternary structure 22

Receptor 31, 38, 4 0 , 4 4 , 4 5 Repressor 57, 65 Ribosomes 58, 59,69, 70, 71, 75 RNA 4 8 , 4 9 , 5 3 , 5 4 , 5 5 , 5 6 , 5 7 , 5 8 , 5 9 , . 65,67,68,69 SCID 94 Second messenger 32, 94 Secondary structure 22 Sickle-cell anaemia 91 Steroids 31 Tertiary structure 22 Thalassaemia 91 Transcription 5 7 , 6 5 , 6 7 , 8 9 Translation 5 9 , 8 9 , 9 1 Ubiquitin 75 X-ray crystallography 1 7 , 1 8 , 8 8

Proteins are of prime importance to the body. Being fundamental to all aspects of cell structure and function, they are the molecular instruments by which the genetic information is expressed. They are present in a bewildering array of shapes and forms, each uniquely specialised for its task. This book takes a closer look at the world of proteins and vividly details their sequences, shapes and functions. Besides reflecting on the perils of both protein malfunction and malnutrition, this slim volume highlights the multifarious roles that proteins play. Dr. Medha S. Rajadhyaksha was awarded Ph.D. in Biophysics by the University of Pune for her work at the Bhabha Atomic Research Centre, Mumbai. Presently a senior faculty of the Life Science Department and Vice-Principal at Sophia College, Mumbai, she is involved in research in Neurobiology. She has written several popular science books, and has contributed a number of articles in newspapers and science magazines. Dr. Sukanya Datta acquired her doctorate in Zoology from the University of Calcutta, before joining the National Institute of Science Communication, New Delhi, where she now works as a scientist. Dr. Datta has been actively involved in popularisation of science, and has authored many popular science books. Besides contributing articles and book reviews in various newspapers and journals, she also writes radio scripts. ISBN 81-237-3105-1 NATIONAL B O O K T R U S T , I N D I A

The Secrets of Proteins

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Microcharacterization of Proteins

Microcharacterization of Proteins

Molecular Modeling of Proteins

Surface Activity of Proteins

Posttranslational Modifications of Proteins

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Anslational Modifications of Proteins

Surface Activity of Proteins

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Modification of Proteins

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The Secrets of Proteins

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Microcharacterization of Proteins

Microcharacterization of Proteins

Molecular Modeling of Proteins

Surface Activity of Proteins

Posttranslational Modifications of Proteins

Production of Recombinant Proteins

Anslational Modifications of Proteins

Surface Activity of Proteins

Proteins of Iron Metabolism

The gluten proteins

Modification of Proteins

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Secrets of the Jedi

Secrets of the Jedi

Secrets of the Dead

The Book of Secrets

The Language of Secrets

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