Single molecule spectroscopy in chemistry physics and biology nobel symposium

Springer Series in

chemical physics

96


Springer Series in

chemical physics
Series Editors: A. W. Castleman, Jr. J. P. Toennies K. Yamanouchi W. Zinth
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Astrid Gr¨aslund
Rudolf Rigler
Jerker Widengren
Editors

Single Molecule Spectroscopy
in Chemistry, Physics
and Biology
Nobel Symposium
With 223 Figures

123

Editors

Professor Astrid Gr¨aslund

Professor Jerker Widengren

Stockholm University
Department of Biophysics
10691 Stockholm, Sweden
E-Mail:

Royal Institute or Technology (KTH)
Department of Biomolecular Physics
10691 Stockholm, Sweden
E-Mail:

Professor Rudolf Rigler
Swiss Federal Institute of Technology Lausanne (EPFL)
1015 Lausanne, Switzerland
E-Mail:

Series Editors:

Professor A.W. Castleman, Jr.
Department of Chemistry, The Pennsylvania State University
152 Davey Laboratory, University Park, PA 16802, USA

Professor J.P. Toennies

Max-Planck-Institut f¨ur Str¨omungsforschung
Bunsenstrasse 10, 37073 G¨ottingen, Germany

Professor K. Yamanouchi
University of Tokyo, Department of Chemistry
Hongo 7-3-1, 113-0033 Tokyo, Japan

Professor W. Zinth
Universit¨at M¨unchen, Institut f¨ur Medizinische Optik
¨
Ottingerstr.
67, 80538 M¨unchen, Germany

Springer Series in Chemical Physics ISSN 0172-6218
ISBN 978-3-642-02596-9
e-ISBN 978-3-642-02597-6
DOI 10.1007/978-3-642-02597-6
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Nobel Symposium, June 2008, at the S˚
anga S¨
aby Conference


Foreword

By selecting the first week of June 2008 for the Nobel Symposium “Single
Molecular Spectroscopy in Chemistry, Physics and Biology”, Rudolf Rigler,
Jerker Widengren and Astrid Gr¨
aslund have once again won the top prize
for Meeting Organizers, providing us with a Mediterranean climate on top of
the warm hospitality that is unique to Sweden. The S˚
anga S¨
aby Conference
Center was an ideal place to spend this wonderful week, and the comfort of
this beautiful place blended perfectly with the high calibre of the scientific
programme. It was a special privilege for me to be able to actively participate
in this meeting on a field that is in many important ways complementary to
my own research. I was impressed by the interdisciplinary ways in which single
molecule spectroscopy has evolved and is currently pursued, with ingredients
originating from physics, all branches of chemistry and a wide range of biological and biomedical research. A beautiful concert by Semmy Stahlhammer
and Johan Ull´en further extended the interdisciplinary character of the symposium. I would like to combine thanks to Rudolf, Jerker and Astrid with a
glance into a future of other opportunities to enjoy top-level science combined
with warm hospitality in the Swedish tradition.
Z¨

urich,
April 2009

Kurt W¨
uthrich


Participants of the Nobel-Symposium 138: First row: Sarah Unterkofler, Anders Liljas, Xiao-Dong Su, Birgitta Rigler, Carlos Bustamante, Toshio Yanagida, Steven Block, Xiaowei Zhuang, Sunney Xie.
Second row: Ivan Scheblykin, Lars Thelander, Petra Schwille, Watt W. Webb, Rudolf Rigler, Jerker Widengren, Peter Lu, Shimon
Weiss, William E Moerner, David Bensimon.
Third row: Anders Ehrenberg, Yu Ming, Fredrik Elinder, Kazuhiko Kinosita, Vladana Vukojevic, Masataka Kinjo, May D Wang, Yu
Ohsugi, Shuming Nie, Andreas Engel, Peter G Wolynes, Michel Orrit, Hans Blom, Johan Hofkens.
Fourth row: Claus Seidel, Heike Hevekerl, Taekjip Ha, Evangelos Sisamakis, Per Ahlberg, Joseph Nordgren, Kurt Wthrich, Sune
Svanberg, Bengt Nordn, Paul Alivisatos, Per Thyberg, Richard Keller, Andriy Chmyrov, Johan Elf, Per Rigler, Kai Hassler, Gustav
Persson, J¨
urgen K¨
ohler, Eric Betzig, Thomas Schmidt, Christoph Br¨
auchle, Elliot Elson, Mans
˙ Ehrenberg, Dimitrios K Papadopoulos,
Ingemar Lundstr¨
om, Horst Vogel, Stefan Wennmalm, Hermann Gaub, H˚
akan Wennerstr¨
om, Yosif Klafter, Julio Fernandez.


Preface

The development of Single Molecule Detection and Spectroscopy started in the
late eighties. The developments came from several areas. Fluorescence-based
single molecule spectroscopy developed in particular from (i) holeburning and

zero phonon spectroscopy of organic molecules at cryo temperatures and (ii)
confocal fluctuation spectroscopy of emitting molecules at elevated temperatures. Of crucial importance for these approaches was the ability to suppress
background radiation to the point where signals of single molecules could be
detected. Today, confocal single molecule analysis is the dominating approach,
particularly in chemistry and in biosciences, but attempts to combine analysis
at low and high temperatures are being pursued.
In parallel with this development, significant progress has been made in
the field of single molecule force spectroscopy. Approaches based on atomic
force microscopy, optical trapping, microneedles or magnetic beads have made
it possible to investigate mechanical properties, and not least, the interplay
between mechanics and chemistry on a single molecule level.
In June 1999 the first Nobel Conference on Single Molecule Spectroscopy
was organized in S¨
odergarn Mansion, Liding¨
o (Sweden) and a comprehensive
presentation of the results obtained in the first decade of single molecule
analysis was given (Orrit, Rigler, Basche (eds.) 1999)
Now after almost another decade, it was of interest to find out whether
the developments and promises presented at the S¨odergarn Conference were
still valid or had even exceeded our expectations.
The contributions to this volume come from the pioneers of the early period
of single molecule spectroscopy as well as from other laboratories which have
made important contributions to demonstrate the importance of SM analysis
in various applications in Chemistry, Physics and BioSciences.
The Nobel Symposium No. 138 dedicated to Single Molecule Spectroscopy
in Chemistry, Physics and Biosciences was held at the Mansion S˚
anga-S¨
aby
situated at the island of Eker¨
o in Lake M¨alaren outside Stockholm, from June

1–6, 2008. The Conference was blessed with pleasant weather and sunshine all
the days. Together with the wonderful surroundings this contributed to many


X

Preface

stimulating opportunities for individual discussions, in parallel with outdoor
excursions including swimming in the lake, jogging tours, walks in the forests
and sauna.
The Symposium started with an evening session on molecules and dynamic
processes by Kurt W¨
uthrich and Martin Karplus. The program of the next
days included the presentation of the fields which initiated single molecule
analysis in cryo temperatures (Moerner,Orrit) followed by confocal analysis
of molecular fluctuations at room temperature (Keller, Rigler, Elson, Webb,
Widengren, Schwille). Major topics in the following sessions included quantum dots (Alivasatos, Nie), the analysis of conformational dynamics (Weiss,
Ha, Seidel), the motion of molecular motors (Yanagida, Kinosita) and replicating assemblies (Bustamante, Block). A special session was devoted to the
analysis of forces operating on single molecules (Gaub, Fernandez) as well as
to high resolution imaging of single molecules (Hell, Betzig, Zhuang, Engel).
Stochastic single molecule events at the cellular level were another important
topic (Xie, Schmidt, Vogel, Wolynes) as well as single molecule enzymology
(Lu, Xie, Rigler, Hofkens, Klafter, K¨
ohler), which together with atomic force
microscopy formed the basis for intense discussions. Several presentations
brought the single molecule methodologies and perspectives to a sub-cellular
and cellular context (Rigler, Schwille, Weiss, Bensimon, Axner, Hell, Betzig,
Zhuang, Schmidt, Xie, Orwar, Br¨
auchle), which seems to form one of several

exciting future directions of this field.
A special event was the evening concert with Semmy Stalhammer on the
violin and Johan Ull´en on the piano. The violin sonata of Cesar Franck and its
masterly performance matched perfectly the level and tension of the scientific
sessions.
As organizers we would like to thank all the invited speakers for their
excellent contributions to this symposium, as well as all those who contributed
with a chapter to this book. We would also like to thank Margareta Klingberg
and colleagues at the conference site of S˚
anga-S¨
aby for the prerequisites and
support of an excellent venue, and not the least the Nobel Foundation for
supporting this Symposium.
Stockholm,
July 2009

Astrid Gr¨
aslund
Rudolf Rigler
Jerker Widengren


Contents

Part I Introductory Lecture: Molecular Dynamics of Single
Molecules
1 How Biomolecular Motors Work: Synergy Between Single
Molecule Experiments and Single Molecule Simulations
Martin Karplus and Jingzhi Pu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

Part II Detection of Single Molecules and Single Molecule
Processes
2 Single-Molecule Optical Spectroscopy and Imaging:
From Early Steps to Recent Advances
William E. Moerner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3 Single Molecules as Optical Probes for Structure and
Dynamics
Michel Orrit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4 FCS and Single Molecule Spectroscopy
Rudolf Rigler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Part III Fluorescence-Correlation Spectroscopy
5 Single-Molecule Spectroscopy Illuminating the Molecular
Dynamics of Life
Watt W. Webb . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
6 Chemical Fluxes in Cellular Steady States Measured by
Fluorescence-Correlation Spectroscopy
Hong Qian and Elliot L. Elson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119


XII

Contents

7 In Vivo Fluorescence Correlation and Cross-Correlation
Spectroscopy
J¨
org M¨

utze, Thomas Ohrt, Zdenˇek Petr´
aˇsek, and Petra Schwille . . . . . . . 139
8 Fluorescence Flicker as a Read-Out in FCS: Principles,
Applications, and Further Developments
Jerker Widengren . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Part IV Quantum Dots and Single Molecule Behaviour
9 Development of Nanocrystal Molecules for Plasmon Rulers
and Single Molecule Biological Imaging
A.P. Alivisatos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
10 Size-Minimized Quantum Dots for Molecular and Cellular
Imaging
Andrew M. Smith, Mary M. Wen, May D. Wang, and Shuming Nie . . . . 187
11 Mapping Transcription Factors on Extended DNA: A
Single Molecule Approach
Yuval Ebenstein, Natalie Gassman, and Shimon Weiss . . . . . . . . . . . . . . . . 203

Part V Molecular Motion of Contractile Elements and Polymer
Formation
12 Single-Molecule Measurement, a Tool for Exploring the
Dynamic Mechanism of Biomolecules
Toshio Yanagida . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
13 Viral DNA Packaging: One Step at a Time
Carlos Bustamante and Jeffrey R. Moffitt . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
14 Chemo-Mechanical Coupling in the Rotary Molecular
Motor F1 -ATPase
Kengo Adachi, Shou Furuike, Mohammad Delawar Hossain, Hiroyasu
Itoh, Kazuhiko Kinosita, Jr., Yasuhiro Onoue, and Rieko Shimo-Kon . . . 271

Part VI Force and Multiparameter Spectroscopy on Functional

Active Proteins
15 Mechanoenzymatics and Nanoassembly of Single
Molecules
Elias M. Puchner and Hermann E. Gaub . . . . . . . . . . . . . . . . . . . . . . . . . . . 289


Contents

XIII

16 Single Cell Physiology
Pierre Neveu, Deepak Kumar Sinha, Petronella Kettunen, Sophie Vriz,
Ludovic Jullien, and David Bensimon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
17 Force-Clamp Spectroscopy of Single Proteins
Julio M Fernandez, Sergi Garcia-Manyes, and Lorna Dougan . . . . . . . . . . 317
18 Unraveling the Secrets of Bacterial Adhesion Organelles
Using Single-Molecule Force Spectroscopy
Ove Axner, Oscar Bj¨
ornham, Micka¨el Castelain, Efstratios Koutris,
Staffan Schedin, Erik F¨
allman, and Magnus Andersson . . . . . . . . . . . . . . . 337

Part VII Nanoscale Microscopy and High Resolution Imaging
19 Far-Field Optical Nanoscopy
Stefan W. Hell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
20 Sub-Diffraction-Limit Imaging with Stochastic Optical
Reconstruction Microscopy
Mark Bates, Bo Huang, Michael J. Rust, Graham T. Dempsey,
Wenqin Wang, and Xiaowei Zhuang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
21 Assessing Biological Samples with Scanning Probes

A. Engel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417

Part VIII Single Molecule Microscopy in Individual Cells
22 Enzymology and Life at the Single Molecule Level
X. Sunney Xie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
23 Controlling Chemistry in Dynamic Nanoscale Systems
Aldo Jesorka, Ludvig Lizana, Zoran Konkoli, Ilja Czolkos, and
Owe Orwar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Part IX Catalysis of Single Enzyme Molecules
24 Single-Molecule Protein Conformational Dynamics in
Enzymatic Reactions
H. Peter Lu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
25 Watching Individual Enzymes at Work
Kerstin Blank, Susana Rocha, Gert De Cremer, Maarten B.J.
Roeffaers, Hiroshi Uji-i, and Johan Hofkens . . . . . . . . . . . . . . . . . . . . . . . . . 495


XIV

Contents

26 The Influence of Symmetry on the Electronic Structure of
the Photosynthetic Pigment-Protein Complexes from Purple
Bacteria
Martin F. Richter, J¨
urgen Baier, Richard J. Cogdell, Silke Oellerich,
and J¨
urgen K¨
ohler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

Part X Fields and Outlook
27 Exploring Nanostructured Systems with Single-Molecule
Probes: From Nanoporous Materials to Living Cells
Christoph Br¨
auchle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
28 Gene Regulation: Single-Molecule Chemical Physics in a
Natural Context
Peter G. Wolynes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561


Contributors

Kengo Adachi
Department of Physics
Faculty of Science and Engineering
Waseda University, Okubo
Shinjuku-ku, Tokyo 169-8555
Japan
A. P. Alivisatos
Department of Chemistry
University of California
Berkeley, USA
and
Materials Science Division
Lawrence Berkeley National Lab
Berkeley, USA

Magnus Andersson
Department of Physics

Ume˚
a University
901 87 Ume˚
a, Sweden
Ove Axner
Department of Physics
Ume˚
a University
901 87 Ume˚
a, Sweden

J¨
urgen Baier
Experimental Physics IV
and Bayreuth Institute for
Macromolecular Research

Universit¨
at Bayreuth
Universtit¨
atsstrasse 30
95440 Bayreuth, Germany
Mark Bates
School of Engineering and Applied
Sciences
29 Oxford Street, Cambridge
MA 02138, USA
David Bensimon
Laboratoire de Physique Statistique
UMR 8550

Ecole Normale Sup´erieure
Paris, France
and
Department of Chemistry and
Biochemistry
University of California at Los
Angeles
Los Angeles, CA, USA
,

Oscar Bj¨
ornham
Department of Applied Physics
and Electronics
Ume˚
a University
901 87 Ume˚
a, Sweden


XVI

Contributors

Kerstin Blank
Department of Chemistry
Katholieke Universiteit Leuven
Leuven, Belgium

Gert De Cremer

Department of Microbial and
Molecular Systems
Katholieke Universiteit Leuven
Leuven, Belgium

Christoph Br¨
auchle
Department of Chemistry und
Biochemistry and Center for
Nanoscience (CeNS)
Ludwig-Maximilians-Universit¨
at
M¨
unchen
Butenandtstrasse 11
81377 M¨
unchen, Germany
Christoph.Braeuchle@
cup.uni-muenchen.de

Ilja Czolkos
Department of Physical Chemistry
Chalmers University of Technology
412 96 Gothenburg, Sweden

Carlos Bustamante
Jason L. Choy Laboratory of Single
Molecule Biophysics and Department
of Physics
University of California, Berkeley

CA 94720, USA
and
Departments of Chemistry and
Molecular and Cell Biology
Howard Hughes Medical Institute
University of California, Berkeley
CA 94720, USA

Micka¨
el Castelain
Department of Physics
Ume˚
a University
901 87 Ume˚
a, Sweden
Richard J. Cogdell
Division of Biochemistry and
Molecular Biology
Institute of Biomedical and Life
Sciences
Biomedical Research Building
University of Glasgow
120 University Place
Glasgow G12 8TA, UK

Graham T. Dempsey
Program in Biophysics
Harvard University, Cambridge
MA 02138, USA
Lorna Dougan

Department of Biological Sciences
Columbia University
New York, NY 10027, USA
Yuval Ebenstein
Department of Chemistry and
Biochemistry
and DOE Institute for Genomics and
Proteomics
UCLA, Germany
Elliot L. Elson
Department of Biochemistry and
Molecular Biophysics
Washington University
St. Louis, MO 63110, USA

A. Engel
Maurice E. M¨
uller Institute for
Structural Biology
Biozentrum, University of Basel
Klingelbergstrasse 70, 4056 Basel
Switzerland
and
Department of Pharmacology
Case Western Reserve University
10900 Euclid Avenue
Wood Bldg 321D, Cleveland
OH 44106, USA

Contributors

Erik F¨
allman
Department of Physics
Ume˚
a University
901 87 Ume˚
a, Sweden
Julio M Fernandez
Department of Biological Sciences
Columbia University, New York
NY 10027, USA

Shou Furuike
Department of Physics
Faculty of Science and Engineering
Waseda University, Okubo
Shinjuku-ku, Tokyo 169-8555
Japan

XVII

Johan Hofkens
Department of Chemistry
Katholieke Universiteit Leuven
Leuven, Belgium

ac.be

Mohammad Delawar Hossain
Department of Physics
Faculty of Science and Engineering
Waseda University, Okubo
Shinjuku-ku, Tokyo 169-8555
Japan
and

Sergi Garcia-Manyes
Department of Biological Sciences
Columbia University
New York, NY 10027, USA

Department of Physics
School of Physical Sciences
Shahjalal University of Science and
Technology
Sylhet-3114, Bangladesh

Natalie Gassman
Department of Chemistry and
Biochemistry
and DOE Institute for Genomics and
Proteomics
UCLA, Germany

Bo Huang
Department of Chemistry and
Chemical Biology
Cornell University, Ithaca

NY, USA

Hermann E. Gaub
Lehrstuhl f¨
ur Angewandte Physik
LMU Munich, Amalienstr. 54
80799 Munich, Germany
and
Center for Nanoscience (CENS)
Nanosystems Initiative Munich
(NIM) and Center for Integrated
Protein Science Munich (CIPSM)
Germany

Stefan W. Hell
Department of NanoBiophotonics
Max Planck Institute for Biophysical
Chemistry
37070 G¨ottingen, Germany
,

and
Howard Hughes Medical Institute
Harvard University, Cambridge
MA 02138, USA

Hiroyasu Itoh
Tsukuba Research Laboratory
Hamamatsu Photonics KK
Tokodai, Tsukuba 300-2635

Japan
Aldo Jesorka
Department of Physical Chemistry
Chalmers University of Technology
412 96 Gothenburg, Sweden


XVIII Contributors

Ludovic Jullien
D´epartement de Chimie UMR 8640
Ecole Normale Sup´erieure
Paris, France

Martin Karplus
Department of Chemistry and
Chemical Biology
Harvard University, Cambridge
MA 02138, USA

Petronella Kettunen
Department of Physiological Science
University of California at Los
Angeles
Los Angeles, CA, USA

Kazuhiko Kinosita, Jr
Department of Physics
Faculty of Science and Engineering
Waseda University, Okubo

Shinjuku-ku, Tokyo 169-8555
Japan


J¨
urgen K¨
ohler
Experimental Physics IV
and Bayreuth Institute for
Macromolecular Research
Universit¨
at Bayreuth
Universtit¨
atsstrasse 30
95440 Bayreuth, Germany

Zoran Konkoli
Microtechnology and Nanoscience
Center
Chalmers University of Technology
412 96 Gothenburg
Sweden

Efstratios Koutris
Department of Physics
Ume˚
a University
901 87 Ume˚
a, Sweden
Ludvig Lizana

Department of Physical Chemistry
Chalmers University of Technology
412 96 Gothenburg, Sweden
H. Peter Lu
Department of Chemistry
Center for Photochemical Sciences
Bowling Green State University
Bowling Green
OH 43403, USA

William E. Moerner
Departments of Chemistry and
(by Courtesy) of Applied Physics
Stanford University, Stanford
CA 94305, USA

Jeffrey R. Moffitt
Jason L. Choy Laboratory of Single
Molecule Biophysics and Department
of Physics
University of California
Berkeley
CA 94720, USA
J¨
org M¨
utze
Biophysics group
Biotechnologisches Zentrum
Technische Universit¨
at Dresden

Tatzberg 47-51
01307 Dresden
Germany,
petra.schwille@
biotec.tu-dresden.de


Contributors

Pierre Neveu
Kavli Institute for Theoretical
Physics
University of California at Santa
Barbara
Santa Barbara
CA, USA

Shuming Nie
Departments of Biomedical
Engineering and Chemistry
Emory University and Georgia
Institute of Technology
101 Woodruff Circle
Suite 2001, Atlanta
GA 30322, USA

Silke Oellerich
Experimental Physics IV
and Bayreuth Institute for
Macromolecular Research

Universit¨
at Bayreuth
Universtit¨
atsstrasse 30
95440 Bayreuth
Germany
Thomas Ohrt
Biophysics group
Biotechnologisches Zentrum
Technische Universit¨
at Dresden
Tatzberg 47-51, 01307 Dresden
Germany,
petra.schwille@
biotec.tu-dresden.de
Yasuhiro Onoue
Department of Physics
Faculty of Science and Engineering
Waseda University, Okubo
Shinjuku-ku, Tokyo 169-8555
Japan
and

XIX

Department of Functional Molecular
Science
The Graduate University for
Advanced Studies (Sokendai)
Okazaki, Aichi 444-8585

Japan
Michel Orrit
MoNOS, LION
Postbox 9504, Leiden University
2300 RA Leiden
The Netherlands

Owe Orwar
Department of Physical Chemistry
Chalmers University of Technology
412 96 Gothenburg, Sweden

Zdenˇ
ek Petr´
aˇ
sek
Biophysics group
Biotechnologisches Zentrum
Technische Universit¨
at Dresden
Tatzberg 47-51
01307 Dresden, Germany
petra.schwille@
biotec.tu-dresden.de
Jingzhi Pu
Laboratoire de Chimie Biophysique
ISIS, Universit´e Louis Pasteur
67000 Strasbourg, France
Elias M. Puchner
Lehrstuhl f¨

ur Angewandte Physik
LMU Munich, Amalienstr. 54
80799 Munich, Germany
and
Center for Nanoscience (CENS)
Nanosystems Initiative Munich
(NIM) and Center for Integrated
Protein Science Munich (CIPSM)
Germany


XX

Contributors

Hong Qian
Department of Applied Mathematics
University of Washington
Seattle, WA 98195, USA
Martin F. Richter
Experimental Physics IV
and Bayreuth Institute for
Macromolecular Research
Universit¨
at Bayreuth
Universtit¨
atsstrasse 30
95440 Bayreuth, Germany
Susana Rocha
Department of Chemistry

Katholieke Universiteit Leuven
Leuven, Belgium
Maarten B. J. Roeffaers
Department of Chemistry
Katholieke Universiteit Leuven
Leuven, Belgium
Michael J. Rust
Department of Physics Harvard
University
Cambridge, MA 02138
USA
Staffan Schedin
Department of Applied Physics and
Electronics
Ume˚
a University
901 87 Ume˚
a, Sweden
Petra Schwille
Biophysics group
Biotechnologisches Zentrum
Technische Universit¨
at Dresden
Tatzberg 47-51, 01307 Dresden
Germany
petra.schwille@
biotec.tu-dresden.de

Rieko Shimo-Kon
Department of Physics

Faculty of Science and Engineering
Waseda University
Okubo, Shinjuku-ku
Tokyo 169-8555, Japan
Deepak Kumar Sinha
Laboratoire de Physique Statistique
UMR 8550
Ecole Normale Sup´erieure
Paris, France

Andrew M. Smith
Departments of Biomedical
Engineering and Chemistry
Emory University and Georgia
Institute of Technology
101 Woodruff Circle
Suite 2001, Atlanta
GA 30322, USA
Hiroshi Uji-i
Department of Chemistry
Katholieke Universiteit Leuven
Leuven, Belgium
Sophie Vriz
Inserm U770
H´emostase et Dynamique Cellulaire
Vasculaire
Le Kremlin-Bicˆetre
France

May D. Wang

Departments of Biomedical
Engineering
Georgia Institute of Technology
313 Ferst Drive
UA Whitaker Building 4106
Atlanta, GA 30332, USA
and


Contributors

Department of Electrical and
Computer Engineering
Georgia Institute of Technology
313 Ferst Drive
UA Whitaker Building 4106
Atlanta, GA 30332, USA
Wenqin Wang
Department of Physics
Harvard University
Cambridge
MA 02138, USA
Watt W. Webb
Cornell University
School of Applied and Engineering
Physics
212 Clark Hall
Ithaca, NY 14853-2501, USA

Shimon Weiss

Department of Chemistry and
Biochemistry
and DOE Institute for Genomics and
Proteomics
UCLA, University of California
Los Angeles, CA, USA

Mary M. Wen
Departments of Biomedical
Engineering and Chemistry
Emory University and Georgia
Institute of Technology
101 Woodruff Circle
Suite 2001, Atlanta
GA 30322, USA
Jerker Widengren
Exp.Biomol.Physics Dept. Appl.
Physics
Royal Institute of Technology (KTH)
Albanova University Center
106 91 Stockholm, Sweden


XXI

Peter G. Wolynes
Department of Chemistry and
Biochemistry
University of California at San Diego
9500 Gilman Drive

La Jolla, CA 92093
USA

X. Sunney Xie
Department of Chemistry and
Chemical Biology
Harvard University
Cambridge
MA 02138 USA

Toshio Yanagida
Graduate School of Frontier
Biosciences
Osaka University, 1-3 Yamadaoka,
Suita, Osaka
565-0871 Japan
and
Formation of soft nano-machines
CREST 1-3 Yamadaoka
Suita, Osaka
565-0871 Japan

osaka-u.ac.jp
.
osaka-u.ac.jp/
Xiaowei Zhuang
Department of Chemistry and
Chemical Biology
Howard Hughes Medical Institute
Harvard University

Cambridge, MA 02138
USA
and
Department of Physics
Program in Biophysics
Harvard University, Cambridge,
MA 02138, USA



Part I

Introductory Lecture: Molecular Dynamics of
Single Molecules


Part II

Detection of Single Molecules and Single
Molecule Processes


1
How Biomolecular Motors Work: Synergy
Between Single Molecule Experiments
and Single Molecule Simulations
Martin Karplus and Jingzhi Pu

Summary. Cells are a collection of machines with a wide range of functions. Most of
these machines are proteins. To understand their mechanisms, a synergistic combination of experiments and computer simulations is required. Some underlying concepts

concerning proteins involved in such machines and their motions are presented. An
essential element is that the conformational changes required for machine function
are built into the structure by evolution. Specific biomolecular motors (kinesin and
F1 −ATPase) are considered and how they work is described.

On the basis of my lecture at Nobel Symposium 138 on Single Molecule Spectroscopy, I shall present studies of proteins that illustrate how single molecule
experiments and single molecule simulations complement each other to provide insights not available from either one by itself. I will focus particularly
on molecular motors and how they work. Before considering specific examples, I shall describe some general properties of the protein free energy surface
and how evolution encodes the required information in protein structures so
that they can perform their motor functions. Figure 1.1a shows a schematic
picture of the free energy of a polypeptide chain under native conditions of
temperature and solvent environment, as a function of an order parameter,
such as the radius of gyration, Rg . We see that at large values of Rg , the
chain has a high free energy and forms what is often referred to as a random coil, though it is now known that, even in a denaturing environment,
there is considerable residual structure. As solution conditions are changed
to stabilize the native state, the coil state condenses to a compact globule.
This can still be disorganized (i.e., no more native structural features than in
the “random” coil) or it can be organized in what is called a molten globule,
which has much of the secondary structural elements (α-helices and β-strands)
of the native protein, but the tertiary structure has not yet formed and the
sidechains are disordered. As Rg continues to decrease, there is usually a
free energy barrier before the collapse to the native state, which is a deep
minimum (on the order of 10 kcal mol−1 ) and narrow on the length scale of
Fig. 1.1a. As the native state is the one in which most, but not all proteins,


4

M. Karplus and J. Pu

energy

(a)

native

globule

(b)

coil

energy

coordinate

coordinate

Fig. 1.1. (a) Schematic free energy surface for a polypeptide that folds to form a
stable protein. The energy is shown as a function of a size coordinate, such as the
radius of gyration Rg . (b) Details of native state energy surface at approximately
constant Rg (see text)

are active, it is useful to examine it at higher resolution. To do so, we choose
a coordinate “perpendicular” to Rg ; by perpendicular we mean that the size
is essentially constant on the scale of Fig. 1.1a. The contributing structures
have very similar values of Rg , but differ in the detailed arrangement of the
atoms, in accord with the fluctuations demonstrated by native state molecular dynamics simulations [1] or measured by X-ray thermal parameters [2].
Figure 1.1b shows that the surface along the perpendicular direction corresponds overall to a “broad” minimum with a complex multiminimum
character. This multiminimum character was demonstrated by quenched

molecular dynamics simulations of myoglobin [3]. They showed that the smallest barriers separating two minima are such that they are crossed in 0.1 ps and
that there is a whole hierarchy of barriers of increasing height that may require
nanosecond, microsecond, or even longer to cross. The quenching simulations
were stimulated by the experiments of Frauenfelder and coworkers [2], who
studied the rebinding of CO to myoglobin after photodissociation over a temperature range of 40–300 K and times ranging from 10−7 to 103 s. What made
such studies possible in an ensemble system is that the photodissociation reaction provides a “trigger”, which synchronizes the initial state of the molecules.
The rebinding reaction was shown to be “complex” [4]; that is, the rebinding reaction is stretched exponential or power law, rather than exponential
in time, and the rate of the reaction decreases faster than expected from the
Arrhenius equation as the temperature is lowered. To interpret both of these
observations Frauenfelder et al. postulated a surface such as that shown in
Fig. 1.1b. The nonexponential time dependence was explained by the ensemble average over myoglobin molecules trapped in different minima, each of


1 How Biomolecular Motors Work

5

which has a different activation energy for rebinding. The non-Arrhenius temperature dependence was rationalized by the “glassy” nature of the protein at
low temperatures. It will be interesting to have single molecule experiments
for myoglobin to confirm the Frauenfelder model.
Recent advances in room-temperature fluorescence spectroscopy have made
possible the real-time observation of single biomolecules, thus circumventing
the problem of synchronization. Of particular interest are distance-sensitive
probes based on fluorescence resonance energy transfer (FRET) [5] or electron
transfer (ET) [6], which provide information on conformational fluctuations.
In the electron transfer experiments I consider here, the Fre/FAD protein
complex was used and the quenching of the fluorescent chromophore FAD by
electron transfer from an excited Tyr was studied. The observed variation in
the quenching rate of a single molecule was interpreted in terms of distance
fluctuations between the FAD and a nearby Tyr, on the basis of the exponential distance dependence of the ET rate [7, 8]. A stretched exponential decay

of the distance autocorrelation function was observed and shown to be consistent with an anomalous diffusion-based model [9, 10]; also, a one-dimensional
generalized Langevin equation (GLE) model with a power-law memory kernel
was found to provide an interpretation of the results [11]. Such formulations
provided compact descriptions of the experiments, but they do not determine
the underlying molecular mechanism that results in the wide distribution of
relaxation times.
There are three tyrosine residues in Fre, Tyr 35, Tyr 72, and Tyr 116, close
to the flavin-binding pocket (Fig. 1.2). Fluorescence lifetime measurements of
the wild-type and mutant Fre/flavin complexes showed that electron transfer
from Tyr 35 to the excited FAD isoalloxazine is responsible for the fluorescence
quenching [6]. The average positions of the bound FAD and the three tyrosine
residues of the protein are shown in the figure. To study the fluctuations in the

Fig. 1.2. Positions of the Tyr residues and FAD in Fre: The average positions of
72
the three nearby Tyr35 , Tyr , and Tyr116 plus FAD in a 5 ns simulation are shown