Single-Molecule Biophysics E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Serie: Advances in Chemical Physics
• Sprache: Englisch

Seitenzahl: 804

Veröffentlichungsjahr: 2011

Beschreibung

Discover the experimental and theoretical developments in optical single-molecule spectroscopy that are changing the ways we think about molecules and atoms The Advances in Chemical Physics series provides the chemical physics field with a forum for critical, authoritative evaluations of advances in every area of the discipline. This latest volume explores the advent of optical single-molecule spectroscopy, and how atomic force microscopy has empowered novel experiments on individual biomolecules, opening up new frontiers in molecular and cell biology and leading to new theoretical approaches and insights. Organized into two parts--one experimental, the other theoretical--this volume explores advances across the field of single-molecule biophysics, presenting new perspectives on the theoretical properties of atoms and molecules. Single-molecule experiments have provided fresh perspectives on questions such as how proteins fold to specific conformations from highly heterogeneous structures, how signal transductions take place on the molecular level, and how proteins behave in membranes and living cells.This volume is designed to further contribute to the rapid development of single-molecule biophysics research. Filled with cutting-edge research reported in a cohesive manner not found elsewhere in the literature, each volume of the Advances in Chemical Physics series serves as the perfect supplement to any advanced graduate class devoted to the study of chemical physics.

Leseprobe

Contents

Cover

Preface to the Series

Title Page

Copyright

Editorial Board

Contributors

Preface

Part 1: Developments on Single-Molecule Experiments

Chapter 1: Staring at a Protein: Ensemble and Single-Molecule Investigations on Protein-Folding Dynamics

I. Introduction

II. Ensemble Investigations of Protein Folding

III. Single-Molecule Investigations of Protein Folding

IV. Summary and Perspective

Acknowledgments

References

Chapter 2: Single-Molecule FRET of Protein-Folding Dynamics

I. Introduction

II. Single-Molecule Spectroscopy

III. Single-Molecule Spectroscopy of Protein Folding

IV. Toward Protein-Folding Mechanisms in the Cell

V. Current Developments and Future Directions

VI. Conclusion

Acknowledgments

References

Chapter 3: Quantitative Analysis of Single-Molecule FRET Signals and its Application to Telomere DNA

I. Introduction

II. FRET Efficiency

III. EFRET for a Single Molecule

IV. Distribution of the FRET Efficiency

V. Advances in FRET Measurements

References

Chapter 4: Force to Unbind Ligand–Receptor Complexes and the Internal Rigidity of Globular Proteins Probed by Single-Molecule Force Spectroscopy

I. Introduction

II. Force Spectroscopy of Ligand–Protein Interactions

III. Internal Rigidity of Protein Molecules

IV. Indentation of Assembled Proteins

V. Future Prospects

References

Chapter 5: Recent Advances in Single-Molecule Biophysics with the Use of Atomic Force Microscopy

I. Introduction

II. Dynamics of Biomolecules

III. Dynamic Studies of Biomolecules with Conventional Techniques

IV. Single-Molecule Methods

V. Relevance of Force in Biology

VI. AFM as a Powerful Technique for Single-Molecule Measurement in Fluids

VII. Force Measurement with AFM: Experimental Setup

VIII. Dynamic Force Spectroscopy for the Study of Conformational Changes of Single Biomolecules at Equilibrium and Far from Equilibrium

IX. Conformational Transformation of Pyranose Rings in Polysaccharides

X. DNA Stretching by AFM

XI. Stretching of Poly(Ethylene Glycol) by AFM

XII. Kinetic Analysis of Conformational Transition Far from Equilibrium Measured by Conventional AFM Methods

XIII. A Single-Molecule Force Spectroscopy Study on the Effect of Temperature on Mechanical Unfolding of the Titin I27 Domain

XIV. Review of Novel Dynamic Force Spectroscopy and Viscoelasticity Measurements of Single Molecules for Biomolecules–Polymers in Fluids with New AFM Techniques

XV. Thermal Noise Spectroscopy for Viscoelastic Measurement with AFM

XVI. Summary

Acknowledgments

References

Chapter 6: Dynamical Single-Molecule Observations of Membrane Protein Using High-Energy Probes

I. Introduction

II. Results and Discussion

III. Conclusion and Future Works

Acknowledgments

References

Chapter 7: Single-Molecular Gating Dynamics for the KcsA Potassium Channel

I. Introduction

II. What Is the Gating of Ion Channels?

III. Structure of the KcsA Channel

IV. Diffracted X-Ray Tracking Method and Related Experiments

V. Conformational Dynamics of the KcsA Channel

VI. Gating Dynamics of the KcsA Channel

VII. Conclusion

Acknowledgments

References

Chapter 8: Static and Dynamic Disorder in IN VITRO Reconstituted Receptor'Adaptor Interaction

I. Introduction

II. Advantages of Kinetic Measurements in in vitro Systems

III. IN VITRO Single-Molecule Measurement of Interactions Between EGFR and Grb2

IV. Kinetics of Interactions Between EGFR and Grb2

V. Conclusion

References

Part 2: Developments on Single-Molecule Theories and Analyses

Chapter 9: Change-Point Localization and Wavelet Spectral Analysis of Single-Molecule Time Series

Introduction

II. General Constraints and Solution Criteria

III. Change-Point Analysis

IV. Wavelet-Correlation Analysis

V. Outlook

References

Chapter 10: Theory of Single-Molecule FRET Efficiency Histograms

I. Introduction

II. FRET Efficiency Histograms

III. Photon Count Rates

IV. Concluding Remarks

References

Chapter 11: Multidimensional Energy Landscapes in Single-Molecule Biophysics

I. Introduction

II. Free Energy Landscape

III. Disconnectivity Graphs

IV. Dimensionality of State-to-State Network and Energy Landscape

V. Effective Free Energy Landscape Extracted from Single-Molecule Time Series

VI. Conclusion and Outlook

Acknowledgments

References

Chapter 12: Generalized Michaelis'Menten Equation for Conformation Modulated Monomeric Enzymes

I. Introduction

II. Model

III. Derivation of the Average Turnover Rate

IV. Classical MM Equation, Detailed Balance, and Zero Conformational Current Condition

V. Non-MM Kinetics

VI. Conclusion

Acknowledgments

Appendix A

Appendix B

Appendix C

References

Chapter 13: Making it Possible: Constructing a Reliable Mechanism from a Finite Trajectory

I. Introduction

II. The RD Forms and Their Relations to the Data and to On–Off KSs

III. Constructing the RD Form From the Data

IV. Concluding Remarks

Appendix A

Appendix B

References

Chapter 14: Free Energy Landscapes of Proteins: Insights from Mechanical Probes

I. Introduction

II. One-Dimensional Models of Mechanical Strength

III. Multidimensionality of Energy Landscapes

IV. Use of the Jarzynski Relation to Directly Determine Free Energies

V. Conclusions

Acknowledgments

References

Chapter 15: Mechanochemical Coupling Revealed by the Fluctuation Analysis of Different Biomolecular Motors

I. Introduction

II. Materials and Methods

III. Results and Discussion

IV. Conclusion

Acknowledgments

References

Author Index

Subject Index

Color Plates

Preface to the Series

Advances in science often involve initial development of individual specialized fields of study within traditional disciplines, followed by broadening and overlapping, or even merging, of those specialized fields, leading to a blurring of the lines between traditional disciplines. The pace of that blurring has accelerated in the last few decades, and much of the important and exciting research carried out today seeks to synthesize elements from different fields of knowledge. Examples of such research areas include biophysics and studies of nanostructured materials. As the study of the forces that govern the structure and dynamics of molecular systems, chemical physics encompasses these and many other emerging research directions. Unfortunately, the flood of scientific literature has been accompanied by losses in the shared vocabulary and approaches of the traditional disciplines, and there is much pressure from scientific journals to be ever more concise in the descriptions of studies, to the point that much valuable experience, if recorded at all, is hidden in supplements and dissipated with time. These trends in science and publishing make this series, Advances in Chemical Physics, a much needed resource.

The Advances in Chemical Physics is devoted to helping the reader obtain general information about a wide variety of topics in chemical physics, a field that we interpret very broadly. Our intent is to have experts present comprehensive analyses of subjects of interest and to encourage the expression of individual points of view. We hope that this approach to the presentation of an overview of a subject will both stimulate new research and serve as a personalized learning text for beginners in a field.

Stuart A. RiceAaron R. Dinner

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Library of Congress Catalog Number: 58-9935

ISBN: 978-1-118-05780-3

oBook ISBN: 978-1-118-13137-4

ePDF ISBN: 978-1-118-13140-4

ePub ISBN: 978-1-118-13138-1

eMobi ISBN: 978-1-118-13139-8

Editorial Board

Moungi G. Bawendi, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Kurt Binder, Condensed Matter Theory Group, Institut für Physik, Johannes Gutenberg-Universitöt Mainz, Mainz, Germany

William T. Coffey, Department of Electronics and Electrical Engineering, Trinity College, University of Dublin, Dublin, Ireland

Karl F. Freed, Department of Chemistry, James Franck Institute, University of Chicago, Chicago, Illinois, USA

Daan Frenkel, Department of Chemistry, Trinity College, University of Cambridge, Cambridge, United Kingdom

Pierre Gaspard, Center for Nonlinear Phenomena and Complex Systems, Universit´ Libre de Bruxelles, Brussels, Belgium

Martin Gruebele, School of Chemical Sciences and Beckman Institute, Director of Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

Jean-Pierre Hansen, Department of Chemistry, University of Cambridge, Cambridge, United Kingdom

Gerhard Hummer, Chief, Theoretical Biophysics Section, NIDDK-National Institutes of Health, Bethesda, Maryland, USA

Ronnie Kosloff, Department of Physical Chemistry, Institute of Chemistry and Fritz Haber Center for Molecular Dynamics, The Hebrew University of Jerusalem, Israel

Ka Yee Lee, Department of Chemistry and The James Franck Institute, The University of Chicago, Chicago, Illinois, USA

Todd J. Martinez, Department of Chemistry, Stanford University, Stanford, California, USA Shaul Mukamel, Department of Chemistry, University of California at Irvine, Irvine, California, USA

Jose Onuchic, Department of Physics, Co-Director Center for Theoretical Biological Physics, University of California at San Diego, La Jolla, California, USA

Steven Quake, Department of Physics, Stanford University, Stanford, California, USA

Mark Ratner, Department of Chemistry, Northwestern University, Evanston, Illinois, USA

David Reichmann, Department of Chemistry, Columbia University, New York, New York, USA

George Schatz, Department of Chemistry, Northwestern University, Evanston, Illinois, USA

Norbert Scherer, Department of Chemistry, James Franck Institute, University of Chicago, Chicago, Illinois, USA

Steven J. Sibener, Department of Chemistry, James Franck Institute, University of Chicago, Chicago, Illinois, USA

Andrei Tokmakoff, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

Donald G. Truhlar, Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, USA

John C. Tully, Department of Chemistry, Yale University, New Haven, Connecticut, USA

Contributors

Rehana Afrin, Innovation Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

AkinoriBaba, Physical Biology Unit, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

Jianshu Cao, Department of Chemistry, MIT, Cambridge, Massachusetts, 02139, USA

Ophir Flomenbom, Flomenbom-BPS, Louis Marshal 19, Tel-Aviv, Israel 62668

Irina V. Gopich, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Atsushi Ikai, Innovation Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan

Masayuki Iwamoto, Department of Molecular Physiology and Biophysics, University of Fukui Faculty of Medical Sciences, 23-3, Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan

Kiyoto Kamagata, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan

Masaru Kawakami, School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan; PRESTO of Japan Science and Technology Corporation (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

Tamiki Komatsuzaki, Molecule & Life Nonlinear Sciences Laboratory, Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita-ku, Sapporo 001-0020, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan

Takashi Konno, Department of Molecular Physiology and Biophysics, University of Fukui Faculty of Medical Sciences, 23-3, Matsuokashimoaizuki, Eiheijicho, Yoshida-gun, Fukui 910-1193, Japan

Miki Morimatsu, Laboratories for Nanobiology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

Daniel Nettels, Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland

Masatoshi Nishikawa, Department of Mathematical and Life Sciences, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

Shigetoshi Oiki, Department of Molecular Physiology and Biophysics, University of Fukui Faculty of Medical Sciences, 23-3, Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan

Kenji Okamoto, Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

Peter D. Olmsted, School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK

Emanuele Paci, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK

Yasushi Sako, Cellular Informatics Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan

Yuji C. Sasaki, Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa City, Chiba, 277-8561, Japan

Benjamin Schuler, Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland

Hiroshi Sekiguchi, Laboratory of Biodynamics, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan; Department of Advanced Materials Science, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa city, Chiba, 277-8561, Japan

Hirofumi Shimizu, Department of Molecular Physiology and Biophysics, University of Fukui Faculty of Medical Sciences, 23-3, Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japan

Attila Szabo, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

Hiroaki Takagi, Department of Physics, Nara Medical University, 840 Shijo-cho, Kashihara Nara 634-8521, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

Satoshi Takahashi, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan

Yukinori Taniguchi, School of Materials Science, Japan Advanced Institute of Science and Technology (JAIST) 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan; Japan Society for the Promotion of Science (JSPS), 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan

Masahide Terazima, Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

Jianlan Wu, Department of Chemistry, MIT, Cambridge, Massachusetts, 02139, USA

Haw Yang, Department of Chemistry, Princeton University, Princeton, NJ 08544, USA

Zu Thur Yew, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK

Preface

Theoretical and experimental breakthroughs are strongly coupled: major advances in fundamental theoretical concepts are often triggered by novel experimental methods and observations. Similarly, new theoretical ideas suggest more experiments. Single-molecule studies are a clear demonstration of this paradigm. The advent of optical single-molecule spectroscopy and atomic force microscopy has empowered novel experiments on individual biomolecules, opening up new frontiers in molecular and cell biology. And these experiments led to new theoretical approaches and insights. The single-molecule approaches offer unique insights not only for the distribution of molecular properties, but also for the dynamics of individual molecules information that cannot be provided by conventional ensemble averaged measurements.

In the past fewyears, important advances have been made in several areas where data from single-molecule experiments have provided fresh new perspectives. Driving these developments are questions including, for example, how proteins fold to specific conformations from the highly heterogeneous structures, how signal transductions take place on the molecular level, as well how proteins behave in membranes and in living cells. A general problem arising from these new experimental observations is the theoretical underpinning for the roles of fluctuations in biochemical reactions. For example: Why biological systems can robustly perform their functions even with the free energy gain or loss of the reactions being comparable to the thermal energy, kBT ?

With a strong conviction that the integration of experimental developments and theoretical advances is essential toward resolving these issues, we have organized two international conferences focusing on identifying and articulating these issues. The first conference was entitled, Linking Single Molecule Spectroscopy and Energy Landscape Perspectives, held on December 3, 2008, at the FUKUOKA convention center, Fukuoka, Japan (organized by T. Komatsuzaki and H. Yang) during the 46th Annual Conference of Biophysical Society of Japan. The second conference was entitled, New Approaches to Complexity of Protein Dynamics by Single Molecule Measurements: Experiments and Theories, from December 7 to 9, 2008, held at the Institute for Protein Research (IPR), Osaka University, Japan (organized by S. Takahashi, T. Komatsuzaki, M. Kawakami).

This volume consists of contributions from participants of the above-mentioned two conferences, including invited speakers, discussants, and organizers. The content of this volume is organized into two parts: one is experimental, and the other is theoretical development on single-molecule biophysics. Part I focuses mainly on three experimental approaches: single-molecule fluorescence based mainly on fluorescence resonance energy transfer (FRET), atomic force microscopy (AFM) and diffracted X-ray tracking (DXT), and their applications to important biological phenomena. This part begins with experimental investigations of protein folding and the development of a new fluorescence method for a long time detection without tethering proteins to glass (Takahashi and Kamagata, Chapter 1) and is followed by conformational and dynamic properties of unfolded proteins based on the confocal detection of single-molecule fluorescence (Nettels and Schuler, Chapter 2), and a quantitative analysis of FRET signals in terms of cumulative distribution functions for telomere DNA (Okamoto and Terazima, Chapter 3). The topic then turns to AFM, which is capable of measuring the mechanical response of a single molecule to an applied force. A systematic AFM study of the mechanical property of the unbinding force of biomolecular complexes and rigidity (stiffness) of various biomolecules are reviewed (Ikai, Afrin, and Sekiguchi, Chapter 4). A novel dynamic force spectroscopy using AFM is discussed, in which a stretched single molecule is driven by an external oscillatory motion and both the static and dynamic mechanical properties of the molecule can be obtained through the deflection of an AFM cantilever (Kawakami and Taniguchi, Chapter 5). A recent breakthrough in single-molecule experiments is the DXT method, which monitors the movements of individual nanocrystals linked to a specific site of proteins at the picometer scale (Sasaki, Chapter 6). The DXT method revealed a twisting motion involved in the gating dynamics of a membrane potassium channel, KcsA (Oiki et al. Chapter 7). Finally, an exploration of complex kinetics at the association/dissociation of epidermal growth factor receptor and Grb2 in cellular signal transductions using an in vitro reconstructed system was reviewed (Takagi, Morimatsu, and Sako, Chapter 8).

Part II focuses on the theoretical progress in single-molecule data analyses. In general, the observed time traces from single molecule are contaminated by several internal noises in addition to external noises arising from experimental settings. For example, the origin of the fluctuation in the FRET data ranges from photophysics, such as blinking and bleaching to different quantum yields of two dye molecules. It is therefore of crucial importance to recognize the existence of two distinct, but complementary stages, in the analyses of single-molecule time series: The first is of extracting the time trace of a desired physical quantity, such as an interdye distance from a noisy signal and the second is of constructing the underlying mechanisms from a scalar time series, such as multidimensional energy landscapes and networks composed of states in a kinetic scheme. Part II includes new theoretical frameworks to extract the scalar time trace of a desired physical quantity buried in a noisy time signal observed in single-molecule experiments, which are designed as an unbiased and quantitative interpretation of the data (Yang, Chapter 9), a comprehensive review of a theory of the FRET efficiency histograms obtained from single-molecule photon counting experiments, which is designed to separate the time trace of photon trajectories in the diffusion process into the distance fluctuation between dye molecules and the other components (Gopich and Szabo, Chapter 10), a new theoretical framework to construct local equilibrium states and the corresponding multidimensional energy landscape from a scalar time series without a priori postulation of the concept of local equilibration and the detailed balance (Baba and Komatsuzaki, Chapter 11), a generalized Michaelis-Menten rate equation for nonequilibrium steady-state turnover reactions in which the original kinetic network model is mapped onto a flux network with unbalanced population currents and the concentration dependence of substrate for the average turnover rate derived (Wu and Cao, Chapter 12), a new construction scheme of a reduced form of the underlying multisubstate kinetic scheme for time trace when composed of two states, where the connections between the nodes in the network can have multiexponentialwaiting time probability density functions (Flomenbom, Chapter 13), a systematic survey of discrepancies found between numerical simulations andAFM experiments for mechanical unfolding of proteins including a caution of oversimplifications due to the projections of the intrinsic multidimensional free energy surface onto a low dimension (Yew, Olmsted, and Paci, Chapter 14), and the exploration of different types of mechanochemical coupling mechanisms (i.e., loose- and tight-coupling scenarios) functioning in myosin II in terms of the mean velocity and velocity fluctuation at various ATP concentration (Takagi and Nishikawa, Chapter 15).

We note, however, that the subject matter included herein should not be regarded as solved; rather, they indicate topics that have been identified for which solutions with various levels of completeness have been provided. It is therefore hoped that the ideas contained in this volume will motivate further innovations in both experiment and theory, and especially their closer interactions in the near future. We finally acknowledge the financial supports for the above two conferences from the following organizations and programs: (1) Japan Science and Technology Agency, Promoting Globalization on Basic Research Programs, (2) Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), and (3) The Institute for Protein Research, Osaka University.

T. KomatsuzakiM. KawakamiS. TakahashiH. YangR. J. Silbey

Part One

Developments on Single-Molecule Experiments

Chapter 1

Staring at a Protein: Ensemble and Single-Molecule Investigations on Protein-Folding Dynamics

Satoshi Takahashi* and Kiyoto Kamagata

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi 980-8577, Japan and CREST, JST Kawaguchi, Saitama 332-0012, Japan

I. Introduction

A. Structural Properties of Folded Proteins

Proteins are natural heteropolymers that possess a remarkable property to fold to their native conformations autonomously, where they perform various physiological functions [1]. Proteins become unfolded in solutions containing high concentrations of denaturants, however, the folded conformations are usually regenerated once the concentration of the denaturants is reduced [2]. This observation demonstrates that the folded conformation is determined by the primary sequence. It further suggests that the folded conformation is predictable. Despite intensive investigations conducted over the past 40 years, the prediction of protein structures still presents an extremely difficult task [3]. In fact, the current situation is that even the prediction of the foldability of the sequences created by single mutation of natural proteins is difficult, reflecting our lack of understanding of the molecular principles governing protein folding. The processes involved in the selection of the folded structures from the unfolded conformations for actual proteins, that is, the dynamic process of protein folding, remains an important subject [4].

Four structural properties of folded proteins distinguish them from other synthetic and biological polymers and from the unfolded proteins. First, proteins are abundant in secondary structures, which are mainly stabilized by hydrogen bonds between main-chain amides. Second, proteins are always compact, and the interior of proteins is packed nearly perfectly, similarly to crystals of organic compounds. Third, the interior of proteins is mostly dehydrated. No water molecule is usually observed in the core domain of proteins. The compactness and the absence of water are related to the fact that the major driving force of protein folding is the hydrophobic interaction. Fourth, the folded conformation of proteins basically consists of a single topology. In contrast, the unfolded proteins possess no secondary structures, and are in expanded conformations with fully solvated polypeptides. The unfolded proteins comprise an astronomical number of heterogeneous conformations that are interconverting into each other. Consequently, the dynamic process of protein folding involves a myriad of molecular events that lead the unfolded proteins to the distinct conformation. The important question persists: How are the four structural properties of the folded proteins organized in the dynamic process of protein folding?

In the past 10 years, we have investigated the dynamics of protein folding based on the four structural properties described above. To detect transient species, we developed time-resolved experimental systems based on rapid solution mixing and ensemble detection [5]. Additionally, we developed a single-molecule fluorescence detection system to monitor the dynamics of individual proteins [6]. In Section II, we summarize our efforts in the ensemble experiments. In Section III, we describe our recent trials in single-molecule experiments. Finally, we offer our perspectives on the investigation of protein-folding dynamics.

B. Cooperativity in the Folding Transitions

Although the unfolded state of a protein comprises an astronomical number of conformations, the observable process of protein folding is surprisingly simple. For many small proteins with chain lengths of <100 residues (small proteins), the folding is cooperative and occurs as the two-state transition from the unfolded state to the native state, implying that the states are separated by an energy barrier (Fig. 1) [7]. The two-state transition is established for many small proteins in both equilibrium and kinetic measurements [8]. Consequently, intermediates other than the unfolded and native states were unidentifiable, even in time-resolved observations, although it is important that the involvement of a small amount of the intermediate was sometimes pointed out in the folding kinetics of small proteins [9]. The cooperativity is a remarkable property of proteins, whose origin bears central importance for elucidation of folding phenomena.