M E T H O D S I N M O L E C U L A R B I O L O G Y

Series Editor John M. Walker

School of Life and Medical Sciences University of Hertfordshire

Hatfield, Hertfordshire , AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Protein Amyloid Aggregation

Methods and Protocols

Edited by

David Eliezer

Weill Cornell Medical College, New York, NY, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2977-1 ISBN 978-1-4939-2978-8 (eBook) DOI 10.1007/978-1-4939-2978-8

Library of Congress Control Number: 2015949999

Springer New York Heidelberg Dordrecht London © Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.

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Editor David Eliezer Weill Cornell Medical College New York , NY , USA

v

This volume is focused on methods for the characterization of aggregation processes that lead to the formation of amyloid fi brils and amyloid oligomers that feature in the etiology of a variety of human disorders collectively known as amyloidoses. The focus includes tech-niques for visualizing early steps on the amyloid formation pathway, methods for capturing and characterizing oligomeric, potentially toxic, intermediates, strategies for preparing and characterizing mature amyloid fi brils, and approaches for understanding templating and transmission of amyloid aggregates. The target audience includes biochemists and bio-physicists with an interest in elucidating the mechanisms of protein amyloid formation, as well as chemists, pharmacologists, and clinicians with an interest in leveraging an under-standing of such mechanisms for the purpose of therapeutic development.

Chapter 1 treats methods to prepare posttranslationally modifi ed amyloid proteins with a focus on the production of phosphorylated forms of the Parkinson’s disease-associated protein alpha-synuclein. Posttranslational modifi cations of synuclein and other amyloid proteins are often associated with disease pathology yet their role in disease etiology has remained unclear, in part because of the diffi culty of producing homogeneously modifi ed proteins for in vitro studies. Chapter 2 describes both chemical synthesis and native chemi-cal ligation strategies for the production of isotopically labeled amyloid proteins for charac-terization by spectroscopic techniques such as two-dimensional infrared spectroscopy or NMR spectroscopy. Detailed procedures are provided for the production of the diabetes- linked amyloidogenic peptide amylin as well as for the amyloid-forming protein alpha-beta crystallin. Chapter 3 describes the use of paramagnetic relaxation enhancement NMR spec-troscopy to detect and describe the earliest interactions between amyloid monomers, using alpha-synuclein as an example, while Chapter 4 describes the use of circular dichroism spectroscopy to detect the presence of helical intermediates during the formation of amy-loid fi brils, in this case using amylin as an example. The formation of helical intermediates has been implicated as a potentially critical step in the formation of fi brillar aggregates by a number of amyloid proteins. Chapter 5 describes innovative applications of fl uorescence correlation spectroscopy to measure oligomer formation both for purifi ed amyloid proteins in vitro and also for fl uorescently labeled amyloid proteins in intact cells, providing a unique approach to observing the amyloid formation process in vivo using huntingtin exon 1 poly-peptides as an example. Chapter 6 describes the application of advanced Raman Spectroscopy methods for the characterization of the process by which amyloid fi brils form, allowing for the characterization of different fi bril regions, such as the core or the surface, as well as for determining the order in which secondary structure is formed during fi bril assembly. The method is illustrated using amyloid formation by lysozyme. Chapter 7 describes an innova-tive use of quantitative electron microscopy to determine the parameters that govern the fi brillization kinetics of the Alzheimer’s protein tau.

Chapters 8 , 9 , and 10 focus on the characterization of oligomeric species formed on the pathway of amyloid fi bril assembly. Chapter 8 describes the use of a powerful combination of mass spectrometry techniques: ion mobility spectrometry and electrospray ionization, in order to characterize the gas phase collision cross sections of different oligomeric species

Pref ace

vi

that are present simultaneously in samples undergoing amyloid fi bril assembly. The heterogeneity of species in such samples has long been a major hurdle to characterizing the fi bril formation process, and this technique is one of very few that is able to provide a simul-taneous analysis and resolution of different species. An application to the aggregation of beta-2- microglobulin is described. Chapter 9 describes techniques to produce stable homo-geneous preparations of oligomers formed by alpha-synuclein as well as a variety of meth-ods to characterize these oligomers, including SDS-PAGE, circular dichroism, electron microscopy, atomic force microscopy, Fourier-transform infrared spectroscopy, and fl uores-cence assays of phospholipid vesicle permeabilization. Chapter 10 also treats the character-ization of oligomers formed from the protein alpha-synuclein but describes the application of a novel single-molecule fl uorescence photobleaching approach to characterizing the number density of the oligomers. Despite intense efforts, reliably determining the distribu-tions of the number of molecules in amyloid oligomers has remained a frustrating chal-lenge, and this method provides a reliable solution to this long-standing issue.

Chapters 11 through 14 describe methods for the characterization of mature amyloid fi brils. Chapter 11 describes protocols for the preparation of alpha-synuclein amyloid fi brils for characterization by solid-state NMR spectroscopy. Solid-state NMR has provided some of our most detailed insights into the structures of amyloid fi brils of a number of proteins and continues to be at the forefront of amyloid fi bril structure determination. Key to the success of this method, however, is the production of high quality samples of fi brils, and this chapter provides an avenue for achieving this. Chapter 12 describes the characteriza-tion of amyloid fi brils formed from the protein tau using electron paramagnetic resonance spectroscopy, which can produce information on local environments within fi brils, provide powerful distance constraints on fi bril conformation, and also distinguish different fi bril populations. Chapter 13 describes the preparation of amyloid fi brils for structure determi-nation by x-ray crystallography. This approach has provided the highest resolution struc-tural views of the central spines of a variety of amyloid-forming sequences. In addition, this method has recently succeeded in resolving the atomic resolution structures of amyloid oligomers, and the preparation of such samples is also described. Chapter 14 describes methods for the analysis of amyloid fi bril structure using amide proton hydrogen exchange monitored by NMR spectroscopy. This approach can provide information, at the single residue level, on solvent accessible regions and hydrogen bonding patterns within fi brils.

Chapters 15 , 16 , and 17 describe computational approaches towards understanding the structure and assembly of amyloid aggregates. This is a rapidly growing area that pro-vides novel insights that are diffi cult or impossible to obtain via experimental methods. Chapter 15 describes a protocol for executing replica exchange molecular dynamics simula-tions of amyloid proteins, using a fragment of the protein tau as an example. Chapter 16 describes the use of molecular dynamics simulations to model the structures of amyloid ion channels, as well as to calculate their ion permeation properties, using the Alzheimer’s amyloid-beta (A-beta) peptide as an example. Ion channel formation by amyloid oligomers is an important potential mechanism for the toxic effects of such species. Chapter 17 describes a protocol for a method that employs Bayesian statistics to leverage experimental data on amyloid proteins for the identifi cation of ensembles of model structures that “best” represent the experimental observables, including statistical parameters to evaluate the signifi cance of various properties of the resulting ensembles.

Chapters 18 , 19 , and 20 describe methods for evaluating processes that may infl uence the toxicity and pathology of amyloid proteins in vivo. Chapter 18 describes experimental methods for evaluating the ability of amyloid species to permeabilize phospholipid bilayers

Preface

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using amylin as an example. Indiscriminant membrane permeabilization by amyloid species has been proposed as a potentially general mechanism for their toxicity. Chapter 19 describes protocols to study the transmission of amyloid species between cells, using the example of alpha-synuclein. Cell-to-cell spread of amyloid species, potentially via a prion-like mecha-nism, has emerged as an area of tremendous interest and may explain observations of how amyloid pathology spreads through the body and brain in neurodegenerative disease. Chapter 20 describes the preparation of amyloid fi brils seeded using material obtained from diseased human or mouse brain tissues in a way that preserves the ultrastructure of the material in the original tissues, using Alzheimer’s brain derived A-beta fi brils as an example. Such samples can then be characterized structurally, in this example using solid-state NMR, in order to delineate the structural basis for different disease-associated fi brillar states and to characterize amyloid strains. The possibility that different amyloid strains, corresponding to different molecular structures of amyloid fi brils, may be associated with different disease presentation and phenotype goes hand-in-hand with the idea that a single or a small num-ber of nucleating aggregation events in the brain or body can lead, via cell-to-cell transmis-sion, to a single or a few dominant fi bril forms.

In summary, this volume presents modern methods and protocols for characterizing amyloid aggregation, amyloid aggregates, and amyloid spread and toxicity from the very earliest manifestations in the form of nucleating conformers or transiently interacting monomers, through the formation of helical intermediates, oligomeric species, membrane- bound oligomers or channels, and fi nally arriving at mature amyloid fi brils, which can be spread from cell to cell, and the molecular details of which may underlie the specifi c features of human disease presentation.

New York, NY, USA David Eliezer

Preface

ix

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I LABELING STRATEGIES

1 Semisynthesis and Enzymatic Preparation of Post-translationally Modified α-Synuclein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Bruno Fauvet and Hilal A. Lashuel

2 Isotope-Labeled Amyloids via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies . . . . . . . . . . . . . . . . . . . . 21 Tianqi O. Zhang , Maksim Grechko , Sean D. Moran , and Martin T. Zanni

PART II KINETICS/MECHANISM

3 Intermolecular Paramagnetic Relaxation Enhancement (PRE) Studies of Transient Complexes in Intrinsically Disordered Proteins . . . . . . . . . . . . . . 45 Maria K. Janowska and Jean Baum

4 Detection of Helical Intermediates During Amyloid Formation by Intrinsically Disordered Polypeptides and Proteins . . . . . . . . . . . . . . . . . . . 55 Andisheh Abedini , Ping Cao , and Daniel P. Raleigh

5 Fluorescence Correlation Spectroscopy: A Tool to Study Protein Oligomerization and Aggregation In Vitro and In Vivo. . . . . . . . . . . . . . . . . . 67 Bankanidhi Sahoo , Kenneth W. Drombosky , and Ronald Wetzel

6 Deep UV Resonance Raman Spectroscopy for Characterizing Amyloid Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Joseph D. Handen and Igor K. Lednev

7 Analyzing Tau Aggregation with Electron Microscopy . . . . . . . . . . . . . . . . . . 101 Carol J. Huseby and Jeff Kuret

PART III OLIGOMERS

8 Characterization of Amyloid Oligomers by Electrospray Ionization-Ion Mobility Spectrometry-Mass Spectrometry (ESI-IMS-MS) . . . . . . . . . . . . . . . 115 Charlotte A. Scarff , Alison E. Ashcroft , and Sheena E. Radford

9 Formation and Characterization of α-Synuclein Oligomers . . . . . . . . . . . . . . . 133 Wojciech Paslawski , Nikolai Lorenzen , and Daniel E. Otzen

10 Fluorescence Methods for Unraveling Oligomeric Amyloid Intermediates . . . . 151 Niels Zijlstra , Nathalie Schilderink , and Vinod Subramaniam

Contents

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PART IV FIBRILS

11 Preparation of Amyloid Fibrils for Magic-Angle Spinning Solid-State NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Marcus D. Tuttle , Joseph M. Courtney , Alexander M. Barclay , and Chad M. Rienstra

12 Spin Labeling and Characterization of Tau Fibrils Using Electron Paramagnetic Resonance (EPR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Virginia Meyer and Martin Margittai

13 Preparation of Crystalline Samples of Amyloid Fibrils and Oligomers. . . . . . . . 201 Asher Moshe , Meytal Landau , and David Eisenberg

14 Quenched Hydrogen Exchange NMR of Amyloid Fibrils . . . . . . . . . . . . . . . . 211 Andrei T. Alexandrescu

PART V COMPUTATIONAL APPROACHES

15 Studying the Early Stages of Protein Aggregation Using Replica Exchange Molecular Dynamics Simulations . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Joan- Emma Shea and Zachary A. Levine

16 Computational Methods for Structural and Functional Studies of Alzheimer’s Amyloid Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Hyunbum Jang , Fernando Teran Arce , Joon Lee , Alan L. Gillman , Srinivasan Ramachandran , Bruce L. Kagan , Ratnesh Lal , and Ruth Nussinov

17 Analyzing Ensembles of Amyloid Proteins Using Bayesian Statistics. . . . . . . . . 269 Thomas Gurry , Charles K. Fisher , Molly Schmidt , and Collin M. Stultz

PART VI TOXICITY AND PATHOLOGY

18 In Vitro Studies of Membrane Permeability Induced by Amyloidogenic Polypeptides Using Large Unilamellar Vesicles . . . . . . . . . . . . . . . . . . . . . . . . 283 Ping Cao and Daniel P. Raleigh

19 Cell Models to Study Cell-to-Cell Transmission of α-Synuclein . . . . . . . . . . . . 291 Eun-Jin Bae , He-Jin Lee , and Seung-Jae Lee

20 Preparation of Amyloid Fibrils Seeded from Brain and Meninges . . . . . . . . . . . 299 Kathryn P. Scherpelz , Jun-Xia Lu , Robert Tycko , and Stephen C. Meredith

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Contents

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ANDISHEH ABEDINI • Diabetes Research Program, Division of Endocrinology, Diabetes and Metabolism , NYU School of Medicine , New York , NY , USA

ANDREI T. ALEXANDRESCU • Department of Molecular and Cell Biology , University of Connecticut , Storrs , CT , USA

FERNANDO TERAN ARCE • Department of Bioengineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA ; Department of Mechanical and Aerospace Engineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA

ALISON E. ASHCROFT • Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology , University of Leeds , Leeds , UK

EUN-JIN BAE • Neuroscience Research Institute and Department of Medicine , Seoul National University College of Medicine , Jongro-gu , South Korea

ALEXANDER M. BARCLAY • Center for Biophysics and Computational Biology , University of Illinois at Urbana-Champaign , Urbana , IL , USA

JEAN BAUM • Department of Chemistry and Chemical Biology , Rutgers University , Piscataway , NJ , USA ; Center for Integrative Proteomics Research , Rutgers University , Piscataway , NJ , USA

PING CAO • Structural Biology Program, Kimmel Center for Biology and Medicine at the Skirball Institute , New York University School of Medicine , New York , NY , USA

JOSEPH M. COURTNEY • Department of Chemistry , University of Illinois at Urbana- Champaign , Urbana , IL , USA

KENNETH W. DROMBOSKY • Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases , University of Pittsburgh School of Medicine , Pittsburgh , PA , USA

DAVID EISENBERG • Department of Biological Chemistry, Howard Hughes Medical Institute (HHMI) , University of California Los Angeles (UCLA) , Los Angeles , CA , USA

BRUNO FAUVET • Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute , Ecole Polytechnique Fédérale de Lausanne , Lausanne , Switzerland

CHARLES K. FISHER • Physics Department , Boston University , Boston , MA , USA ALAN L. GILLMAN • Department of Bioengineering, Materials Science Program , University

of California, San Diego , La Jolla , CA , USA MAKSIM GRECHKO • Department of Chemistry , University of Wisconsin-Madison , Madison ,

WI , USA THOMAS GURRY • Computational and Systems Biology Initiative , Massachusetts Institute of

Technology , Cambridge , MA , USA ; Research Laboratory of Electronics , Massachusetts Institute of Technology , Cambridge , MA , USA

JOSEPH D. HANDEN • Department of Chemistry , University at Albany, SUNY , Albany , NY , USA

CAROL J. HUSEBY • Interdisciplinary Biophysics Graduate Program, Department of Molecular and Cellular Biochemistry , The Ohio State University College of Medicine , Columbus , OH , USA

Contributors

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HYUNBUM JANG • Cancer and Infl ammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research , National Cancer Institute at Frederick , Frederick , MD , USA

MARIA K. JANOWSKA • Department of Chemistry and Chemical Biology , Rutgers University , Piscataway , NJ , USA

BRUCE L. KAGAN • Department of Psychiatry, David Geffen School of Medicine, Semel Institute for Neuroscience and Human Behavior , University of California , Los Angeles , CA , USA

JEFF KURET • Department of Molecular and Cellular Biochemistry , The Ohio State University College of Medicine , Columbus , OH , USA

RATNESH LAL • Department of Bioengineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA ; Department of Mechanical and Aerospace Engineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA

MEYTAL LANDAU • Department of Biology , Technion-Israel Institute of Technology , Haifa , Israel

HILAL A. LASHUEL • Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute , Ecole Polytechnique Fédérale de Lausanne , Lausanne , Switzerland ; Qatar Biomedical Research Institute , Hamad Bin Khalifa University , Qatar Foundation , Doha , Qatar

IGOR K. LEDNEV • Department of Chemistry , University at Albany, SUNY , Albany , NY , USA

HE-JIN LEE • Institute of Biomedical Science and Technology , Konkuk University , Seoul , South Korea ; Department of Anatomy, School of Medicine , Konkuk University , Seoul , South Korea

JOON LEE • Department of Mechanical and Aerospace Engineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA

SEUNG-JAE LEE • Neuroscience Research Institute and Department of Medicine , Seoul National University College of Medicine , Jongro-gu , South Korea

ZACHARY A. LEVINE • Department of Chemistry and Biochemistry , University of California Santa Barbara , Santa Barbara , CA , USA ; Department of Physics , University of California Santa Barbara , Santa Barbara , CA , USA

NIKOLAI LORENZEN • Department of Molecular Biology, Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) , Aarhus University , Aarhus C , Denmark ; Department of Protein Biophysics and Formulation , Novo Nordisk A/S , Måløv , Denmark

JUN-XIA LU • Laboratory of Chemical Physics, NIDDK , National Institutes of Health , Bethesda , MD , USA

MARTIN MARGITTAI • Department of Chemistry and Biochemistry , University of Denver , Denver , CO , USA

STEPHEN C. MEREDITH • Department of Pathology , Biochemistry and Molecular Biology , The University of Chicago , Chicago , IL , USA

VIRGINIA MEYER • Department of Chemistry and Biochemistry , University of Denver , Denver , CO , USA

SEAN D. MORAN • Department of Chemistry , University of Wisconsin-Madison , Madison , WI , USA

ASHER MOSHE • Department of Biology , Technion-Israel Institute of Technology , Haifa , Israel

Contributors

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RUTH NUSSINOV • Cancer and Infl ammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research , National Cancer Institute at Frederick , Frederick , MD , USA ; Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine , Tel Aviv University , Tel Aviv , Israel

DANIEL E. OTZEN • Department of Molecular Biology, Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) , Aarhus University , Aarhus , Denmark

WOJCIECH PASLAWSKI • Department of Molecular Biology, Center for Insoluble Protein Structures (inSPIN), Interdisciplinary Nanoscience Center (iNANO) , Aarhus University , Aarhus C , Denmark ; Department of Clinical Neuroscience , Karolinska Institutet , Stockholm , Sweden

SHEENA E. RADFORD • Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology , University of Leeds , Leeds , UK

DANIEL P. RALEIGH • Department of Chemistry , Stony Brook University , Stony Brook , NY , USA ; Graduate Program in Biochemistry and Structural Biology , Stony Brook University , Stony Brook , NY , USA

SRINIVASAN RAMACHANDRAN • Department of Bioengineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA ; Department of Mechanical and Aerospace Engineering, Materials Science Program , University of California, San Diego , La Jolla , CA , USA

CHAD M. RIENSTRA • Department of Chemistry , University of Illinois at Urbana- Champaign , Urbana , IL , USA ; Department of Biochemistry , University of Illinois at Urbana-Champaign , Urbana , IL , USA ; Center for Biophysics and Computational Biology , University of Illinois at Urbana-Champaign , Urbana , IL , USA

BANKANIDHI SAHOO • Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases , University of Pittsburgh School of Medicine , Pittsburgh , PA , USA

CHARLOTTE A. SCARFF • Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology , University of Leeds , Leeds , UK

KATHRYN P. SCHERPELZ • Department of Biochemistry and Molecular Biology , The University of Chicago , Chicago , IL , USA

NATHALIE SCHILDERINK • Nanobiophysics, MESA+ Institute for Nanotechnology, Faculty of Science and Technology , University of Twente , Enschede , The Netherlands ; Nanobiophysics, MIRA Institute for Biomedical Technology and Technical Medicine , University of Twente , Enschede , The Netherlands

MOLLY SCHMIDT • Research Laboratory of Electronics , Massachusetts Institute of Technology , Cambridge , MA , USA ; Department of Electrical Engineering and Computer Science , Massachusetts Institute of Technology , Cambridge , MA , USA

JOAN-EMMA SHEA • Department of Chemistry and Biochemistry , University of California Santa Barbara , Santa Barbara , CA , USA ; Department of Physics , University of California Santa Barbara , Santa Barbara , CA , USA

COLLIN M. STULTZ • Computational and Systems Biology Initiative , Massachusetts Institute of Technology , Cambridge , MA , USA ; Research Laboratory of Electronics , Massachusetts Institute of Technology , Cambridge , MA , USA ; Department of Electrical Engineering and Computer Science , Massachusetts Institute of Technology , Cambridge , MA , USA ;

Contributors

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The Institute for Medical Engineering and Science , Massachusetts Institute of Technology , Cambridge , MA , USA

VINOD SUBRAMANIAM • FOM Institute AMOLF , Amsterdam , The Netherlands ; Nanobiophysics, MESA+ Institute for Nanotechnology, Faculty of Science and Technology , University of Twente , Enschede , The Netherlands ; Nanobiophysics, MIRA Institute for Biomedical Technology and Technical Medicine , University of Twente , Enschede , The Netherlands

MARCUS D. TUTTLE • Department of Chemistry , University of Illinois at Urbana- Champaign , Urbana , IL , USA

ROBERT TYCKO • Laboratory of Chemical Physics, NIDDK , National Institutes of Health , Bethesda , MD , USA

RONALD WETZEL • Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases , University of Pittsburgh School of Medicine , Pittsburgh , PA , USA

MARTIN T. ZANNI • Department of Chemistry , University of Wisconsin-Madison , Madison , WI , USA

TIANQI O. ZHANG • Department of Chemistry , University of Wisconsin-Madison , Madison , WI , USA

NIELS ZIJLSTRA • FOM Institute AMOLF , Amsterdam , The Netherlands ; Nanobiophysics, MESA+ Institute for Nanotechnology, Faculty of Science and Technology , University of Twente , Enschede , The Netherlands

Contributors