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biological and medical physics, biomedical engineering

biological and medical physics, biomedical engineering

The fields of biological and medical physics and biomedical engineering are broad, multidisciplinary and dynamic. They lie at the crossroads of frontier research in physics, biology, chemistry, and medicine. The Biological and Medical Physics, Biomedical Engineering Series is intended to be comprehensive, covering a broad range of topics important to the study of the physical, chemical and biological sciences. Its goal is to provide scientists and engineers with textbooks, monographs, and reference works to address the growing need for information.

Books in the series emphasize established and emergent areas of science including molecular, membrane, and mathematical biophysics; photosynthetic energy harvesting and conversion; information processing; physical principles of genetics; sensory communications; automata networks, neural networks, and cellular automata. Equally important will be coverage of applied aspects of biological and medical physics and biomedical engineering such as molecular electronic components and devices, biosensors, medicine, imaging, physical principles of renewable energy production, advanced prostheses, and environmental control and engineering.

Editor-in-Chief:

Elias Greenbaum, Oak Ridge National Laboratory,

Oak Ridge, Tennessee, USA

Editorial Board:

Masuo Aizawa, Department of Bioengineering,

Tokyo Institute of Technology, Yokohama, Japan

Olaf S. Andersen, Department of Physiology,

Biophysics & Molecular Medicine,

Cornell University, New York, USA

Robert H. Austin, Department of Physics, Princeton University, Princeton, New Jersey, USA

James Barber, Department of Biochemistry,

Imperial College of Science, Technology

and Medicine, London, England

Howard C. Berg, Department of Molecular

and Cellular Biology, Harvard University,

Cambridge, Massachusetts, USA

Victor Bloomf ield, Department of Biochemistry, University of Minnesota, St. Paul, Minnesota, USA

Robert Callender, Department of Biochemistry,

Albert Einstein College of Medicine,

Bronx, New York, USA

Britton Chance, Department of Biochemistry/

Biophysics, University of Pennsylvania,

Philadelphia, Pennsylvania, USA

Steven Chu, Department of Physics,

Stanford University, Stanford, California, USA

Louis J. DeFelice, Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

Johann Deisenhofer, Howard Hughes Medical

Institute, The University of Texas, Dallas,

Texas, USA

George Feher, Department of Physics,

University of California, San Diego, La Jolla,

California, USA

Hans Frauenfelder, CNLS, MS B258,

Los Alamos National Laboratory, Los Alamos,

New Mexico, USA

Ivar Giaever, Rensselaer Polytechnic Institute,

Troy, New York, USA

Sol M. Gruner, Department of Physics, Princeton University, Princeton, New Jersey, USA

Judith Herzfeld, Department of Chemistry,

Brandeis University, Waltham, Massachusetts, USA

Mark S. Humayun, Doheny Eye Institute,

Los Angeles, California, USA

Pierre Joliot, Institute de Biologie Physico-Chimique, Fondation Edmond de Rothschild, Paris, France

Lajos Keszthelyi, Institute of Biophysics, Hungarian Academy of Sciences, Szeged, Hungary

Robert S. Knox, Department of Physics

and Astronomy, University of Rochester, Rochester,

New York, USA

Aaron Lewis, Department of Applied Physics,

Hebrew University, Jerusalem, Israel

Stuart M. Lindsay, Department of Physics

and Astronomy, Arizona State University,

Tempe, Arizona, USA

David Mauzerall, Rockefeller University,

New York, New York, USA

Eugenie V. Mielczarek, Department of Physics

and Astronomy, George Mason University, Fairfax,

Virginia, USA

Markolf Niemz, Klinikum Mannheim,

Mannheim, Germany

V. Adrian Parsegian, Physical Science Laboratory,

National Institutes of Health, Bethesda,

Maryland, USA

Linda S. Powers, NCDMF: Electrical Engineering, Utah State University, Logan, Utah, USA

Earl W. Prohofsky, Department of Physics, Purdue University, West Lafayette, Indiana, USA

Andrew Rubin, Department of Biophysics, Moscow

State University, Moscow, Russia

Michael Seibert, National Renewable Energy

Laboratory, Golden, Colorado, USA

David Thomas, Department of Biochemistry,

University of Minnesota Medical School,

Minneapolis, Minnesota, USA

Samuel J. Williamson, Department of Physics, New York University, New York, New York, USA

J. Fitter T. Gutberlet J. Katsaras

Neutron Scattering

in Biology

Techniques and Applications

With 240 Figures

123

Dr. Jorg¨ Fitter

Dr. John Katsaras

Forschungszentrum Julich¨ GmbH

National Research Council

Abt. IBI-2

 

Chalk River

52425 Julich,¨

Germany

K0J 1J0 Ontario, Canada

e-mail: j.fitter@fz-juelich.de

e-mail: john.katsaras@nrc.gc.ca

Dr. Thomas Gutberlet

Laboratory of Neutron Scattering Paul Scherrer Institut

5232 Villigen, Switzerland e-mail: thomas.gutberlet@psi.ch

Library of Congress Control Number: 2005934097

ISSN 1618-7210

ISBN-10 3-540-29108-3 Springer Berlin Heidelberg New York

ISBN-13 978-3-540-29108-4 Springer Berlin Heidelberg New York

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specif ically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microf ilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law.

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The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specif ic statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

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Preface

“Certainly no subject or field is making more progress on so many fronts at the present moment, than biology, and if we were to name the most powerful assumption of all, which leads one on and on in an attempt to understand life, it is that all things are made of atoms, and that everything that living things do can be understood in terms of the jigglings and wigglings of atoms.”

Richard P. Feynmann, from “Six easy pieces” (1963)

In 1932, James Chadwick discovered the neutron, but initially the only sources of neutrons were from the radioactive decay of unstable nuclei. It was not until 1942 when Enrico Fermi constructed the first nuclear reactor in the squash courts beneath the University of Chicago’s Stagg Field, that a controlled and sustained nuclear chain reaction was achieved. After World War II, nuclear reactors became available for civilian research, and in 1945 Ernest Wollan set up a double-crystal di ractometer at ORNL’s Graphite Reactor. This marks the beginning of neutron scattering.

Neutrons produced by present reactorand accelerator-based sources, typically have wavelengths in the order of ˚Angstroms, and hence are wellsuited for probing the structures and motions of molecules. For biological materials rich in hydrogen, the large di erence in scattering cross-sections between hydrogen and deuterium provides the possibility of contrast variation, a powerful method achieved by selective deuteration for emphasizing, or not, the scattering from a particular portion of a molecule or molecular assembly. Using a variety of scattering methods, the structures and dynamics of biological systems can be determined.

The present compilation aims to provide the reader with some of the important applications of neutron scattering in structural biology, biophysics, and systems relevant to biology.

The location of hydrogen atoms in biomolecules such as, proteins, is – despite the high brilliance and power of third generation synchrotron

VI Preface

sources – not readily available by X-ray crystallography or related physical techniques. In the case of hydrogens attached to electronegative atoms (e.g., O and N), even high resolution X-ray structures (resolution <1 ˚A) cannot unequivocally locate these H atoms. On the other hand, these atoms can e ectively be located using high resolution crystallographic neutron di raction methods. Radiation damage leading to changes in metal oxidation state and subsequent loss of hydrogens can also pose a problem with X-rays, but not so with neutrons. When good quality, large (>1 mm3) single crystals cannot be obtained, low resolution neutron di raction o ers an alternative technique in determining the hydrated structure of macromolecules and their various hydrogen-bonding patterns.

Small-angle neutron scattering (SANS) is probably the technique most often applied to biological materials as it can probe the size, shape and conformation of macromolecules and macromolecular complexes in aqueous solution on a length scale from ten to several thousand ˚Angstroms. The ability to scatter from materials in solution allows for biologically relevant conditions to be mimicked, and also permits for the study of samples that are either di cult or impossible to crystallize. In recent years, SANS has greatly benefited from the production of “cold neutrons” that have wavelengths 10–20 times larger than “thermal neutrons”, allowing SANS to examine complex materials, such as living cells.

Over the past decade, neutron reflectometry has increasingly become an important technique for the characterization of biological and biomimetic thin films attached to a solid support, in contact with water. Advancements in sample environments, instrumentation, and data analysis now make it possible to obtain high resolution information about the composition of these materials along the axis perpendicular to the plane of the membrane or substrate. Most recently, a newly developed phase-sensitive neutron reflectometry technique also allows direct inversion of the reflectometry data to obtain unique compositional depth profiles of the films in question.

Studies exploring the relationship between the function and the dynamics of biological systems are still in their nascent stages. Incoherent neutron scattering (INS) techniques such as, elastic (EINS), quasielastic (QINS), and inelastic (IINS) neutron scattering, along with molecular dynamics (MD) simulations o er the real possibility of investigating the dynamics associated with a molecule’s biological function(s). Using the large incoherent scattering crosssection intrinsic to naturally abundant hydrogen atoms, various INS type measurements can be carried out. These results, in conjunction with MD simulations, o er a glimpse of for example, a protein’s internal structure on the picosecond time scale. Moreover, the current developments of intense pulsed neutron sources promise, in the near future, to accelerate our understanding of the relationship between a molecule’s dynamics and its function.

The study of materials under di cult environmental conditions (such as high magnetic fields, high pressures, shear, and 100% relative humidity) is by no means straight forward and requires specialized equipment. In many

Preface VII

cases, these experiments are better accommodated by the fact that neutrons interact weakly, thus nondestructively, with many commonly used materials (e.g., aluminum and its alloys) that are readily available and suitable for the construction of sample environments. The conditions created by these specialized environments provide us with a more detailed physical understanding of biologically relevant materials.

The present volume begins with a general introduction into the generation and properties of neutrons and is followed by a series of papers describing the various elastic and inelastic neutron scattering techniques used to study biological and biologically relevant systems. The reader is introduced to the basic principles of neutron crystallography, low resolution neutron di raction, neutron small-angle scattering, neutron reflectometry, inelastic and quasielastic neutron scattering, and neutron spin echo spectroscopy. Papers describing sample environments and preparatory techniques, in addition to molecular dynamics simulations used to evaluate the neutron data, are also included. Finally, there are a series of papers describing recent neutron research that has elucidated the structure and dynamics of soluble proteins, membrane embedded proteins, and of complex biological aggregates.

The editors wish to express their great appreciation to all of the contributors whose diligence, e orts, and timeliness made this compilation possible.

J¨ulich

J¨org Fitter

Villigen

Thomas Gutberlet

Chalk River

John Katsaras

Spring 2005

 

Contents

1 Neutron Scattering for Biology

 

T.A. Harroun, G.D. Wignall, J. Katsaras . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

1.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

1.2

Production of Neutrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

1.3

Elements of Neutron Scattering Theory . . . . . . . . . . . . . . . . . . . . . . .

5

 

1.3.1

Properties of Neutrons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

 

1.3.2 Energy and Momentum Transfer . . . . . . . . . . . . . . . . . . . . . . .

5

 

1.3.3

Di raction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6

 

1.3.4 Scattering Length and Cross-Section . . . . . . . . . . . . . . . . . . . .

7

 

1.3.5 Coherent and Incoherent Cross-Sections . . . . . . . . . . . . . . . . .

8

1.4

Neutron Di raction and Contrast . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10

 

1.4.1

Contrast and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11

 

1.4.2

Contrast and Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

 

1.4.3

Contrast and Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13

1.5

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16

References . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

17

Part I Elastic Techniques

2 Single Crystal Neutron Di raction

and Protein Crystallography

C.C. Wilson, D.A. Myles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.2Single Crystal Neutron Di ractometers:

Basic Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.2.1 Development of Single Crystal Neutron Di ractometers . . . 25

2.2.2Achievements of Neutron Macromolecular

Crystallography at Reactor Sources . . . . . . . . . . . . . . . . . . . . . 25 2.2.3 Developments at Spallation Sources . . . . . . . . . . . . . . . . . . . . 28

XContents

2.2.4Forward Look for Instrumentation

for Neutron Macromolecular Crystallography . . . . . . . . . . . . 29 2.2.5 Improvements in Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2.3 Information from Neutron Crystallography . . . . . . . . . . . . . . . . . . . . 32 2.3.1 Neutron Crystallography of Molecular Materials . . . . . . . . . 32 2.3.2 Neutron Crystallography in Structural Biology . . . . . . . . . . . 33

2.3.3Sample and Data Requirements

for Single Crystal Neutron Di raction . . . . . . . . . . . . . . . . . . 34

2.4Brief Review of the Use of Neutron Di raction

in the Study of Biological Structures . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.4.1 Location of Hydrogen Atoms . . . . . . . . . . . . . . . . . . . . . . . . . . 36 2.4.2 Solvent Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.4.3 Hydrogen Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.4.4 Low Resolution Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.4.5 Other Biologically Relevant Molecules . . . . . . . . . . . . . . . . . . 39

2.5 Recent Developments and Future Prospects . . . . . . . . . . . . . . . . . . . . 41 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3 Neutron Protein Crystallography:

Hydrogen and Hydration in Proteins

N. Niimura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.2 Complementarity of Neutrons and X-rays . . . . . . . . . . . . . . . . . . . . . . 44

3.2.1 Refinement of Hydrogen Positions . . . . . . . . . . . . . . . . . . . . . . 44

3.2.2Hydrogen Atoms Which Cannot be Predicted

Stereochemically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.3 Hydrogen Bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 3.3.1 Weak and Strong Hydrogen Bonding . . . . . . . . . . . . . . . . . . . 50 3.3.2 Bifurcated Hydrogen Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

3.4 H/D Exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 3.5 Hydration in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.5.1 Experimental Observation of Hydration Molecules . . . . . . . . 55 3.5.2 Classification of Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 3.5.3 Dynamic Behavior of Hydration . . . . . . . . . . . . . . . . . . . . . . . . 58

3.6 Crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.7 Conclusions and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4 Neutron Protein Crystallography: Technical Aspects and Some Case Studies at Current Capabilities and Beyond

M. Blakeley, A.J.K. Gilboa, J. Habash, J.R. Helliwell, D. Myles,

J. Raftery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 4.2 Data Collection Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64