Biology at the Single-Molecule Level

Biology at the Single Molecule Level

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Biology at the Single Molecule Level

Edited by

S.H. Leuba National Cancer Institute, NIH, Bethesda, MD, USA and J. Zlatanova Department of Chemistry and Chemical Engineering Polytechnic University Brooklyn, New York, NY, USA

2001 PERGAMON AMSTERDAM - LONDON - NEW YORK - OXFORD - PARIS - SHANNON - TOKYO

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CONTENTS Editorial Single-molecule biochemistry coming of age S. Leuba and J. Zlatanova

VII

Parti (reprinted from Progress in Biophysics & Molecular Biology 74 / 1-2) Protein structural dynamics by single-molecule fluorescence polarization J,N. Forkey, M.E, Quinlan and Y.E. Goldman

1

Single molecule force spectroscopy in biology using the atomic force microscope /. Zlatanova, SM. Lindsay and S.H. Leuba

37

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering M. Carrion-VasqueZy A.F. Oberhausen T.E. Fisher, PE, Marszalek, H. Li and J.M, Fernandez

63

The importance of molecular structure and conformation: learning with scanning probe microscopy B,L. Smith

93

Twisting and stretching single DNA molecules T. Stricky J.-F Allemand, V. Croquette and D. Bensimon

115

Part II (reprinted from Progress in Biophysics & Molecular Biology 77/1) Single molecule measurements of titin elasticity K. Wang, J.G. Forbes and A J, Jin

1

Analyses of single-molecule mechanical recordings: application to actomyosin interactions A.E. Knight, C. Veigel, C. Chambers and J.E. Molloy

45

Structural and functional imaging with carbon nanotube AFM probes LH. Hafner, C-L Cheung, A.T Woolley and CM. Lieber

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Editorial

Single-molecule biochemistry coming of age

Nowadays, we are witnessing a true revolution in the way we can study the structure and function of biological macromolecules. The advent of instrumentation and techniques that allow us to investigate the dynamic behavior of single macromolecules or their complexes in real time has given birth to a whole new field: single-molecule biochemistry. We have attempted to compile a group of reviews that would cover most of the significant developments in the new field, both with respect to the technical advances in instrumentation, and the kinds of results that may be obtained by studying and/or manipulating single molecules or macromolecular complexes. The range of topics is by no means exhaustive, but it may serve as an introduction to this new and quickly developing field. The large number of papers could not be accommodated in one issue, hence the two issues. It was our intent to give the authors the full freedom to not only review the field, but also to express their own views and speculations in a way that is difficult, sometimes impossible, in original experimental papers. The authors wiUingly made use of this freedom; the stringent reviews of the manuscripts that followed was only meant to make sure that the presentation of the facts was correct, with little impingement on this freedom. We hope that the readers will be the beneficiaries of this editorial approach. Finally, we would like to mention that a similar compendium of single-molecule biochemistry reviews appeared as a minireview series in the Journal of Biological Chemistry in 1999; the prologue to that series by the series editor Kensal van Holde coveys all the excitement of the new field (K. van Holde, J. Biol. Chem. 274, 1999, 14515). S. Leuba, J. Zlatanova Department of Chemical Engineering, Chemistry and Materials Science Polytechnic University, Six Metro Tech Center Brooklyn, NY 11201, USA E-mail address: [email protected]

VII

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Biology at the Single Molecule Level S.H. Leuba and J. Zlatanova (Eds) © 2001 Elsevier Science Ltd. All rights reserved

Review

Protein structural dynamics by single-molecule fluorescence polarization Joseph N. Forkey*, Margot E. Quinlan, Yale E. Goldman School of Medicine, University of Pennsylvania, Physiology Department, Pennsylvania Muscle Institute, D700 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6083, USA

Contents 1.

Introduction 1.1. Protein rotational motions 1.2. Fluorescence polarization 1.3. Single-molecule fluorescence polarization 1.4. Early single-molecule fluorescence experiments

2.

Experimental methods 2.1. Fluorescent probes 2.2. Instrumentation 2.2.1. Far-field excitation 2.2.2. Total internal reflection excitation 2.2.3. Near-field optical probe excitation 2.2.4. Microscope objectives and emission optics 2.2.5. Detectors

8 9 11 12 13 14 14 16

3.

Analysis of single-molecule fluorescence polarization data 3.1. Stationary fluorophores 3.1.1. Absorption polarization ratios 3.1.2. Emission polarization ratios 3.2. Non-stationary molecules 3.2.1. Fast wobble 3.2.2. Slow wobble 3.3. Measurement of the axial angle, 6

16 17 17 18 19 20 21 21

4.

Applications

23

5.

Conclusions

27

^Corresponding author.

2 2 5 6 7

Acknowledgements

28

Appendix A. Dependence of polarized fluorescence intensity on molecular orientation A.l. Excitation of a single fluorophore A.2. Fluorescence from a single fluorophore

28 28 30

References

31

Keywords: Single molecule; Fluorescence; Polarization; Evanescent wave; Microscopy; Bifunctional; Dynamics; Total internal reflection

1. Introduction The astonishing progress made during the last two decades toward understanding the machinery of cells has been fueled in large part by developments in structural biology, molecular biology, and biophysics. Thousands of protein structures have been solved at atomic resolution and the amino acid sequences and roles of many specific motifs are now known. While application of these techniques has greatly enhanced our understanding of the structure and biochemistry of proteins, elucidation of the quantitative relationships between structural changes in the proteins, enzymatic activity, signaling, energy conversion and macromolecular interactions will require further development of novel methods. In this chapter we describe a new technique, single-molecule fluorescence polarization (SMFP), that has the potential to fill a void in conventional methods for determining the structural basis of protein activity. Using state-of-the-art optical microscopes and novel fluorescent probes attached to specific protein domains, SMFP makes it possible to quantify angular rotations that mediate function in individual protein molecules. To demonstrate the importance of protein domain rotational motions, this chapter begins with several examples of biophysical systems in which orientational changes are essential to their mechanism. This is followed by a discussion of the advantages of detecting such rotations with individual molecules and a brief synopsis of the development of SMFP and early experiments. The next two sections are devoted to technical considerations, including characteristics of extrinsic fluorescent probes, optical apparatus and data analysis. Finally, recent work using this technique is reviewed. 1.1. Protein rotational motions Crystallographic studies of proteins trapped in several different configurations analogous to states of the functioning system often lead to hypotheses for the structural basis of the catalytic activity. They also identify Hkely sites of interaction with upstream and downstream functional partners. Distinct conformations are typically characterized by diff'erences in the relative orientations of nearby compact domains Hnked by hinges or swivels composed of glycine residues or flexible loops. The impHed rotations have direct bearing on the functional output as illustrated here by several examples where large orientation changes have been discovered. Due to the hehcal structure of double-stranded DNA (dsDNA), nucleic acid processing enzymes produce a torque when unwinding the helix to gain access to the base sequence. These enzymes catalyze processes such as replication, transcription, repair and recombination.

Topoisomerase II removes accumulated strain in dsDNA and enables separation of chromosomes or untangling of circular DNA after replication. It operates by passing one dsDNA segment through a 'gate' temporarily created in another double-stranded segment. During passage of the intact dsDNA through the gate, quaternary structural changes in the DNA-opening platform take place. Dramatic reorganization of accessory protein domains include rotations of up to 170° (Fig. la; Pass et al., 1999). During protein biosynthesis, two GTP-binding elongation factors (EF-Tu and EF-G) enable the ribosome to accurately translate the genetic code into amino acid sequences and to maintain the codon reading frame. In each elongation cycle of a nascent polypeptide, an aminoacylated transfer RNA (tRNA) binds to the ribosome as a ternary complex with EF-Tu and GTP. If the tRNA's anti-codon matches the codon in the template messenger RNA, EF-Tu hydrolyzes the GTP and dissociates. The amino acid is then transferred from the tRNA to the peptide. Crystal structures show a nearly 90° rotation between domains in EF-Tu accompanying GTP hydrolysis (Fig. lb; Clark et a l , 1999). The tRNA also rotates -60° relative to the ribosome. EF-G participates in translocation of the mRNA and two tRNA molecules by exactly 3 bases along the synthetic machinery. Whether EF-G functions as a 'motor' and whether it exhibits large domain rotations are controversial (Rodnina et al., 1999). The F Q - F I ATP synthase couples energy from a proton gradient across the inner mitochondrial membrane into phosphorylation of ADP to form ATP. This process is the major end product of mitochondrial respiration. FQ is thought to be a motor that rotates a protein driveshaft (the y subunit; Walker, 1994; Boyer, 1997). Torque transmitted to the Fi domain drives the 'uphill' ATP synthetic reaction. When uncoupled from FQ, F I hydrolyzes ATP and rotates the y subunit in the opposite direction (Yasuda et al., 1998). Myosin, the motor protein in muscle contraction and in many types of non-muscle motihty, translocates along the cytoskeletal filament, actin. In crystal structures of myosin with various nucleotide analogs bound, the angle between the region containing the actin and nucleotide binding sites (the motor domain) and that containing the myosin Hght chains varies by up to 70° (Fig. Ic; Rayment et al., 1993a,b; Dominguez et al., 1998; Houdusse et al., 1999; Corrie et al., 1999). These studies and other data suggest that during the enzymatic ATPase cycle, a tilting of the light chain domain amplifies sub-nanometer structural changes at the nucleotide binding site into several nanometers of motion (Goldman, 1998). Voltage-gated ion channels underhe electrical impulses in the surface membranes of excitable cells such as neurons and muscle fibers. N a ^ , K"^ and Ca^"^ channels are all composed of homologous repeated domains that form a membrane-spanning pore. The channels are normally closed when transmembrane voltage is negative inside of the cell relative to the extracellular space, but they open when the potential decreases or reverses. The fourth membrane spanning segment (S4) within each domain contains positively charged residues and is thought to serve as the voltage sensor. X-ray crystallography of ion channels has been more difficult than for soluble proteins, but the structure of a homologous bacterial K^ channel (without voltage gating) has recently been solved (Doyle et al., 1998). While this structure has resulted in many new insights, the motions associated with gating are still unknown. Experiments using fluorescent probes attached to S4 segments of eukaryotic K^ channels, however, suggest that the voltage sensor either translates or tilts when the membrane electrical field is altered (Mannuzzu et al., 1996; Yang et al., 1996; Loots and Isacoff^, 1998).

Fig. 1. Examples of crystal structures showing protein domain rotations, (a) Topoisomerase II (T2). Both the Rossman fold (red) and the B' hook (orange) domains rotate relative to the DNA binding platform (blue) during activity. (Reprinted from with permission from Pass et al., 1999, Structural Biology, Nature 6, 322-326.) (b) Ribosomal elongation factor-Tu undergoes a large nucleotide-hydrolysis-dependent reorientation of domains. Note the relative positions of domain I (yellow) versus domains II and III (green) and the transition from a-helix to p-strand in Switch I (red). (Reprinted from FEBS Letters 452, Clark et al., "Structural information for explaining the molecular mechanism of protein biosynthesis" pp. 41-46 © 1999 with permission from Elsevier Science.) (c) Crystal structure of chicken skeletal muscle myosin subfragment-1 (red, blue, tan, and magenta) docked to actin (gray, brown and green; Rayment et al., 1993a). The angle of the light chain domain has been modified to show the relative orientation change determined fromfluorescencepolarization experiments with bifunctional rhodamine (see Section 2.1) when a muscle fiber is suddenly allowed to shorten during isometric contraction. (Reprinted from Corrie et al. "Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction". Nature 400, 425^30 © 1999 with permission from Nature (http://www.nature.com/).

In all of these systems, plausible relationships have been proposed between the observed structural changes and the functional output. Many aspects, though, are unresolved. These include the kinetics of the events, the relationship to ATP or GTP hydrolysis and how each protein is controlled by the chemical, mechanical and topological states of its functional partners. Thus testing the structurally derived hypotheses requires detection of the timing and extent of the motions during activity. 1.2. Fluorescence polarization While X-ray crystallography provides remarkable spatial resolution of long-lived, static states, it provides no temporal information on functionally relevant motions except in rare instances (e.g. Schlichting et al., 1990). Conclusions about the mechanism of activity must usually be inferred from the structures of a few trapped states. Furthermore, even these configurations are approximations of the native structures since the environment within a crystal is much different from the cytoplasm or membrane of a cell. In particular, the solvents and intermolecular forces within a crystal are usually unphysiological. Entire macromolecular complexes do not often crystalhze, so the structural data are usually limited to subcomplexes, individual subunits or even products of proteolytic cleavage. Nuclear magnetic resonance (NMR) also yields atomic spatial resolution and may be performed under aqueous conditions, often closer to a native environment. However, this technique is limited to small proteins (e, t)il/T)Q-^'/'^P,{e^,

0 , , e)Pe(0e, (/>e, Oc) dQ^dQ^dt

(6)

(see also Cantor and Schimmel, 1980, Section 8-2). Here, p(0a,