Visit https://ebookultra.

com to download the full version and

explore more ebooks

Allostery Methods and Protocols 1st Edition

James K. Kranz

_____ Click the link below to download _____

https://ebookultra.com/download/allostery-methods-and-
protocols-1st-edition-james-k-kranz/

Explore and download more ebooks at ebookultra.com

Here are some suggested products you might be interested in.
Click the link to download

Haemostasis Methods and Protocols 1st Edition Anthony K.

C. Chan

https://ebookultra.com/download/haemostasis-methods-and-protocols-1st-
edition-anthony-k-c-chan-2/

Haemostasis Methods and Protocols 1st Edition Anthony K.

C. Chan

https://ebookultra.com/download/haemostasis-methods-and-protocols-1st-
edition-anthony-k-c-chan/

G Protein Signaling Methods and Protocols 1st Edition

Wendy K. Greentree

https://ebookultra.com/download/g-protein-signaling-methods-and-
protocols-1st-edition-wendy-k-greentree-2/

Disease Gene Identification Methods and Protocols 1st

Edition Johanna K. Distefano

https://ebookultra.com/download/disease-gene-identification-methods-
and-protocols-1st-edition-johanna-k-distefano/
Plant Cold Acclimation Methods and Protocols 1st Edition
Dirk K. Hincha

https://ebookultra.com/download/plant-cold-acclimation-methods-and-
protocols-1st-edition-dirk-k-hincha/

G Protein Signaling Methods and Protocols 1st Edition

Wendy K. Greentree

https://ebookultra.com/download/g-protein-signaling-methods-and-
protocols-1st-edition-wendy-k-greentree/

Molecular Chaperones Methods and Protocols Methods in

Molecular Biology v787 2011th Edition Stuart K. Calderwood

https://ebookultra.com/download/molecular-chaperones-methods-and-
protocols-methods-in-molecular-biology-v787-2011th-edition-stuart-k-
calderwood/

Viral Vectors for Gene Therapy Methods and Protocols 1st

Edition James N. Warnock

https://ebookultra.com/download/viral-vectors-for-gene-therapy-
methods-and-protocols-1st-edition-james-n-warnock/

Protein Expression in Mammalian Cells Methods and

Protocols 1st Edition James L. Hartley (Auth.)

https://ebookultra.com/download/protein-expression-in-mammalian-cells-
methods-and-protocols-1st-edition-james-l-hartley-auth/
Allostery Methods and Protocols 1st Edition James K.
Kranz Digital Instant Download
Author(s): James K. Kranz, José C. Clemente (auth.), Aron W. Fenton (eds.)
ISBN(s): 9781617793332, 1617793337
Edition: 1
File Details: PDF, 6.61 MB
Year: 2012
Language: english
METHODS MOLECULAR BIOLOGY
TM
IN

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
.
 Allostery
Methods and Protocols

Edited by

Aron W. Fenton
Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center,
Kansas City, KS, USA
Editor
Aron W. Fenton
Department of Biochemistry and Molecular Biology
The University of Kansas Medical Center
Kansas City, KS, USA
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-333-2 e-ISBN 978-1-61779-334-9
DOI 10.1007/978-1-61779-334-9
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011938679

# Springer ScienceþBusiness Media, LLC 2012

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
Printed on acid-free paper

Humana press is a part of Springer Science+Business Media (www.springer.com)

Dedication

I am convinced that all that is in the Universe revolves around my amazing wife; without
her efforts, I could not do science. I have been in the fortunate position to have been
trained by three mentors who are not only good people, but also believe in science at the
highest caliber. Therefore, I would like to dedicate this book to these four individuals:
Shellee Fenton and Drs. James B. Blair, Gregory D. Reinhart, and Gerald M. Carlson.

v
Preface

In the past 7 years, allostery has resurfaced as a major focus in understanding protein
structure/function. Much of this rejuvenated interest has been driven by the ability of
NMR to monitor protein dynamics and the potential of determining how these dynamics
contribute to protein functions, including allostery (1–6). A second driving force for the
recent interest is a growing appreciation that allosteric drugs offer safety advantages over
conventional drugs (7–9). This renewed interest has resulted in several reviews on allostery
(10–13).
At the onset of any discussion on allostery, it is beneficial to review the exact phenome-
non included in the discussion. Shortly after the original use of “allosteric” (14), confusion
over the definition of this term showed up in the literature. One source of confusion is
whether “allostery” and “cooperative” should be treated as two synonyms to describe the
same principle or if these words describe two different phenomena. To indicate similarities
at the phenomenological level, it is now common to use “allostery” and “cooperative”
interchangeably, with further definition as either “homotropic,” to indicate energetic
coupling when the two ligands are identical, or “heterotropic,” to indicate energetic
coupling when the two ligands are nonidentical. Even with these distinctions, the classifi-
cation of homotropic and heterotropic as independent forms of regulation has been much
debated. Alberto Sols articulated why these properties should be considered as related but
independent properties by emphasizing that homotropic mechanisms require that the
protein is an oligomer (15):
. . .because of confusion between two frequently linked but essentially independent concepts: (i)
specifically regulatory sites and (ii) multiplicity of interacting equal sites in oligomeric proteins. . ..
To compound the tendency to confusion, oligomerism is not only not necessarily linked to allosteric
(heterotropic) effects but is not even the only basis for positive cooperativity (homotropic). . ..

By contrast, a purely thermodynamic view led Harvey Fisher and coworkers to express
the similarities in these two properties (16):
The term “cooperativity,” or, more precisely, “heterotropic cooperativity” has been used occasionally
to describe systems such as that shown . . . (in an allosteric energy cycle). . . in cases where the binding of
one ligand either increases or decreases the affinity of a second, chemically distinguishable ligand.
A majority of workers in the field, however, prefer to restrict the use of the term “cooperativity” to
homotropic systems, and to refer to such effects in heterotropic systems as “positive and negative
interactions.” . . ..however, such a formal distinction between homotropic and heterotropic systems
(implying as it does that the two classes of systems require totally different mechanisms to achieve what
is essentially the same result) is an unwarranted assumption and one which may prove to be misleading.

Given this long standing historical debate, we have found the most productive
approach is to define the type of regulation that is being described. However, one distinc-
tion that should be noted is the additional challenges associated with the study of homo-
tropic systems since the concentrations of the two ligands cannot be varied independently.
In this book, the majority of the chapters focus on studies of heterotropic systems.
However, given the historical association between heterotropic and homotropic effects,
techniques specific to the study of homotropic systems are also represented.

vii
viii Preface

A second level of confusion is whether “allostery” includes any reference to a change in

protein conformation. The original definition given by Monod et al. in 1963 (14) had no
reference to conformational changes. Shortly thereafter, Monod and coworkers offered
a plausible model to explain allostery derived from assumed conformational changes (17).
The 1965 reference has been used to suggest that the recent introduction of dynamics into
the discussion of allostery offers a “new view” of allostery (3, 18, 19). Others have relied on
the 1963 definition to emphasize that the original definition of allostery placed no con-
straints on the molecular source of allosteric regulation and that dynamics were always
accounted for in the description of this phenomena (11, 12, 20, 21).
In the Fenton laboratory, we use the word “allostery” to refer to heterotropic coupling
events, with no implication that the mechanism for this through-protein communication is
restricted to a change in protein conformation. Therefore, allosteric regulation is defined
functionally as how a macromolecule binds one ligand differently when a second ligand is
or is not prebound to the macromolecule. Since the definition of allostery influences what
is expected as the “molecular source of allostery” (22), the use of the same definition has
been strongly encouraged throughout all chapters in this volume (12). However, unifying
the use of terms across all structure/function studies is an unrealistic goal, and even in
several chapters of this volume, the influence of historical deviations of our favored
definition is apparent.
Despite the semantic debates regarding classification, the common feature of allosteric
systems is ligand-induced, through-protein changes. Therefore, any technique that can be
used to study protein structure/function questions can be applied to the study of allostery.
As such, the primary value of this book is the logic that is necessary to study this
phenomenon, a phenomenon that is well recognized through the history of the life
sciences and very poorly understood at the molecular level.

Kansas City, KS, USA Aron W. Fenton

References

1. Tzeng, S. R., and Kalodimos, C. G. Protein dynamics and allostery: an NMR view, Curr Opin Struct
Biol.
2. Smock, R. G., and Gierasch, L. M. (2009) Sending signals dynamically, Science 324, 198–203.
3. Kern, D., and Zuiderweg, E. R. (2003) The role of dynamics in allosteric regulation, Curr Opin Struct
Biol 13, 748–757.
4. Lipchock, J. M., and Loria, J. P. Nanometer propagation of millisecond motions in V-type allostery,
Structure 18, 1596–1607.
5. Bruschweiler, S., Schanda, P., Kloiber, K., Brutscher, B., Kontaxis, G., Konrat, R., and Tollinger, M.
(2009) Direct observation of the dynamic process underlying allosteric signal transmission, J Am
Chem Soc 131, 3063–3068.
6. Yan, J., Liu, Y., Lukasik, S. M., Speck, N. A., and Bushweller, J. H. (2004) CBFbeta allosterically
regulates the Runx1 Runt domain via a dynamic conformational equilibrium, Nat Struct Mol Biol 11,
901–906.
7. Peracchi, A., and Mozzarelli, A. (2010) Exploring and exploiting allostery: Models, evolution, and
drug targeting, Biochim Biophys Acta.
8. Groebe, D. R. (2006) Screening for positive allosteric modulators of biological targets, Drug discovery
today 11, 632–639.
9. Groebe, D. R. (2009) In search of negative allosteric modulators of biological targets, Drug discovery
today 14, 41–49.
10. Hilser, V. J. Biochemistry. An ensemble view of allostery, Science 327, 653–654.
 Preface ix

11. Reinhart, G. D. (2004) Quantitative analysis and interpretation of allosteric behavior,

Methods Enzymol 380, 187–203.
12. Fenton, A. W. (2008) Allostery: an illustrated definition for the ‘second secret of life’, Trends Biochem
Sci 33, 420–425.
13. Lindsley, J. E., and Rutter, J. (2006) Whence cometh the allosterome?, Proc Natl Acad Sci U S A 103,
10533–10535.
14. Monod, J., Changeux, J. P., and Jacob, F. (1963) Allosteric proteins and cellular control systems,
J Mol Biol 6, 306–329.
15. Sols, A. (1981) Multimodulation of Enzyme Activity, Current Topics in Cellular Regulation 19,
77–101.
16. Subramanian, S., Stickel, D. C., Colen, A. H., and Fisher, H. F. (1978) Thermodynamics of hetero-
tropic interactions. The glutamate dehydrogenase . NADPH . glutamate complex, J Biol Chem 253,
8369–8374.
17. Monod, J., Wyman, J., and Changeux, J. P. (1965) On the Nature of Allosteric Transitions: a Plausible
Model, J Mol Biol 12, 88–118.
18. Gunasekaran, K., Ma, B., and Nussinov, R. (2004) Is allostery an intrinsic property of all dynamic
proteins?, Proteins 57, 433–443.
19. Swain, J. F., and Gierasch, L. M. (2006) The changing landscape of protein allostery, Curr Opin Struct
Biol 16, 102–108.
20. Weber, G. (1972) Ligand binding and internal equilibria in proteins, Biochemistry 11, 864–878.
21. Cui, Q., and Karplus, M. (2008) Allostery and cooperativity revisited, Protein Sci 17, 1295–1307.
22. Fenton, A. W., Johnson, T. A., and Holyoak, T. (2010) The pyruvate kinase model system, a
cautionary tale for the use of osmolyte perturbations to support conformational equilibria in allostery,
Protein Sci 19, 1796–1800.
Acknowledgment

This work was supported in part by NIH grant DK78076.

xi
Contents

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Acknowledgment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

PART I MONITORING ALLOSTERIC FUNCTION

1 Binding Techniques to Study the Allosteric Energy Cycle . . . . . . . . . . . . . . . . . . . . . . 3

James K. Kranz and José C. Clemente
2 Kinetic Trapping of a Key Hemoglobin Intermediate . . . . . . . . . . . . . . . . . . . . . . . . . 19
Jo M. Holt and Gary K. Ackers
3 Allosteric Coupling Between Transition Metal-Binding Sites
in Homooligomeric Metal Sensor Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Nicholas E. Grossoehme and David P. Giedroc
4 Studying the Allosteric Energy Cycle by Isothermal Titration Calorimetry. . . . . . . . 53
Marta Martinez-Julvez, Olga Abian, Sonia Vega, Milagros Medina,
and Adrian Velazquez-Campoy
5 Detecting “Silent” Allosteric Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Harvey F. Fisher
6 Using Mutant Cycle Analysis to Elucidate Long-Range Functional
Coupling in Allosteric Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Jai A.P. Shanata, Shawnalea J. Frazier, Henry A. Lester,
and Dennis A. Dougherty

PART II MONITORING ALLOSTERIC CONFORMATIONAL CHANGES

7 A Review of Methods Used for Identifying Structural Changes

in a Large Protein Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Owen W. Nadeau and Gerald M. Carlson
8 Allosteric Mechanisms of G Protein-Coupled Receptor Signaling:
A Structural Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Tarjani M. Thaker, Ali I. Kaya, Anita M. Preininger,
Heidi E. Hamm, and T.M. Iverson
9 Dynamic Light Scattering to Study Allosteric Regulation . . . . . . . . . . . . . . . . . . . . . . 175
Aaron L. Lucius, P. Keith Veronese, and Ryan P. Stafford
10 Dissecting the Linkage Between Transcription Factor Self-Assembly
and Site-Specific DNA Binding: The Role of the Analytical Ultracentrifuge. . . . . . . 187
Amie D. Moody, James P. Robblee, and David L. Bain
11 Fluorescence Correlation Spectroscopy and Allostery: The Case of GroEL . . . . . . . 205
Gabriel A. Frank, Amnon Horovitz, and Gilad Haran
12 The Morpheein Model of Allostery: Evaluating Proteins
as Potential Morpheeins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Eileen K. Jaffe and Sarah H. Lawrence
xiii
xiv Contents

PART III MONITORING ALLOSTERIC CHANGES IN PROTEIN

DYNAMICS/SUBPOPULATION DISTRIBUTION
13 Combining NMR and Molecular Dynamics Studies for Insights
into the Allostery of Small GTPase–Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . 235
Liqun Zhang, Sabine Bouguet-Bonnet, and Matthias Buck
14 Hydrogen–Deuterium Exchange Study of an Allosteric Energy Cycle. . . . . . . . . . . . 261
Dorothy Beckett
15 Ensemble Properties of Network Rigidity Reveal Allosteric Mechanisms. . . . . . . . . 279
Donald J. Jacobs, Dennis R. Livesay, James M. Mottonen,
Oleg K. Vorov, Andrei Y. Istomin, and Deeptak Verma

PART IV MACROMOLECULAR AND LIGAND ENGINEERING

ALLOSTERIC FUNCTIONS

16 An In Vivo Approach to Isolating Allosteric Pathways

Using Hybrid Multimeric Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Cuijuan Tie and Gregory D. Reinhart
17 Mutations in the GABAA Receptor that Mimic
the Allosteric Ligand Etomidate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Stuart A. Forman and Deirdre Stewart
18 Allosteric Regulation of Human Liver Pyruvate Kinase by Peptides
that Mimic the Phosphorylated/Dephosphorylated N-Terminus. . . . . . . . . . . . . . . . 335
Charulata B. Prasannan, Qingling Tang, and Aron W. Fenton
19 In Silico-Screening Approaches for Lead Generation: Identification
of Novel Allosteric Modulators of Human-Erythrocyte Pyruvate Kinase. . . . . . . . . . 351
Ashutosh Tripathi and Martin K. Safo
20 Identification of Allosteric-Activating Drug Leads for Human
Liver Pyruvate Kinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Aron W. Fenton

PART V COMPUTATIONAL METHODS/AIDS IN THE STUDY OF ALLOSTERY

21 A Critical Evaluation of Correlated Mutation Algorithms

and Coevolution Within Allosteric Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Dennis R. Livesay, Kyle E. Kreth, and Anthony A. Fodor
22 The Advantage of Global Fitting of Data Involving
Complex Linked Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Petr Herman and J. Ching Lee
23 Predicting Binding Sites by Analyzing Allosteric Effects. . . . . . . . . . . . . . . . . . . . . . . . 423
Dengming Ming and Michael E. Wall

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Contributors

OLGA ABIAN • Institute of Biocomputation and Physics of Complex Systems (BIFI),

Universidad de Zaragoza, Zaragoza, Spain; Departamento de Bioquı́mica y Biologı́a
Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain;
Fundación ARAID, Diputación General de Aragón, Aragón, Spain
GARY K. ACKERS • (Deceased) Department of Biochemistry & Molecular Biology,
Washington University School of Medicine, St. Louis, MO, USA
DAVID L. BAIN • Department of Pharmaceutical Sciences, University of Colorado,
Aurora, CO, USA
DOROTHY BECKETT • Department of Chemistry and Biochemistry,
University of Maryland, College Park, MD, USA
SABINE BOUGUET-BONNET • Methodologie RMN (CRM2; UMR 7036, UHP-CNRS),
Faculté des Sciences et Techniques, Nancy-Université, Vandoeuvre-les-Nancy, France
MATTHIAS BUCK • Department of Physiology and Biophysics, Case Comprehensive Cancer
Center, Center for Proteomics and Bioinformatics, Case Western Reserve University
School of Medicine, Cleveland, OH, USA; Department of Neurosciences,
Case Comprehensive Cancer Center, Center for Proteomics and Bioinformatics,
Case Western Reserve University School of Medicine, Cleveland, OH, USA;
Department of Pharmacology, Case Comprehensive Cancer Center, Center for
Proteomics and Bioinformatics, Case Western Reserve University School of Medicine,
Cleveland, OH, USA
GERALD M. CARLSON • Department of Biochemistry and Molecular Biology,
University of Kansas Medical Center, Kansas City, KS, USA
JOSÉ C. CLEMENTE • Oncology Research & Development, GlaxoSmithKline,
Upper Providence, PA, USA
DENNIS A. DOUGHERTY • Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA, USA
ARON W. FENTON • Department of Biochemistry and Molecular Biology,
The University of Kansas Medical Center, Kansas City, KS, USA
HARVEY F. FISHER • Department of Biochemistry and Molecular Biology,
University of Kansas Medical Center, Kansas City, KS, USA
ANTHONY A. FODOR • Department of Bioinformatics and Genomics,
University of North Carolina at Charlotte, Charlotte, NC, USA
STUART A. FORMAN • Department of Anesthesia Critical Care & Pain Medicine,
Massachusetts General Hospital, Boston, MA, USA
GABRIEL A. FRANK • Departments of Structural Biology and Chemical Physics,
Weizmann Institute of Science, Rehovot, Israel
SHAWNALEA J. FRAZIER • Division of Biology, California Institute of Technology,
Pasadena, CA, USA
DAVID P. GIEDROC • Department of Chemistry, Indiana University, Bloomington,
IN, USA

xv
xvi Contributors

NICHOLAS E. GROSSOEHME • Department of Chemistry, Indiana University,

Bloomington, IN, USA; Department of Chemistry, Physics and Geology,
Winthrop University, Rock Hill, SC, USA
HEIDI E. HAMM • Department of Pharmacology, Vanderbilt University Medical Center,
Nashville, TN, USA
GILAD HARAN • Department of Chemical Physics, Weizmann Institute of Science,
Rehovot, Israel
PETR HERMAN • Faculty of Mathematics and Physics, Institute of Physics,
Charles University, Prague, Czech Republic
JO M. HOLT • Oro Valley, AZ, USA
AMNON HOROVITZ • Department of Structural Biology, Weizmann Institute of Science,
Rehovot, Israel
ANDREI Y. ISTOMIN • Department of Bioinformatics and Genomics,
University of North Carolina at Charlotte, Charlotte, NC, USA
T.M. IVERSON • Departments of Biochemistry and Pharmacology, Vanderbilt University
Medical Center, Nashville, TN, USA
DONALD J. JACOBS • Department of Physics and Optical Science, University of North
Carolina at Charlotte, Charlotte, NC, USA
EILEEN K. JAFFE • Fox Chase Cancer Center, Philadelphia, PA, USA
ALI I. KAYA • Department of Pharmacology, Vanderbilt University Medical Center,
Nashville, TN, USA
JAMES K. KRANZ • Biopharmaceuticals Research & Development, GlaxoSmithKline,
Upper Merion, PA, USA
KYLE E. KRETH • Department of Bioinformatics and Genomics, University of North
Carolina at Charlotte, Charlotte, NC, USA
SARAH H. LAWRENCE • Fox Chase Cancer Center, Philadelphia, PA, USA
J. CHING LEE • Department of Biochemistry and Molecular Biology, University of Texas
Medical Branch, Galveston, TX, USA
HENRY A. LESTER • Division of Biology, California Institute of Technology,
Pasadena, CA, USA
DENNIS R. LIVESAY • Department of Bioinformatics and Genomics,
University of North Carolina at Charlotte, Charlotte, NC, USA
AARON L. LUCIUS • Department of Chemistry, The University of Alabama
at Birmingham, Birmingham, AL, USA
MARTA MARTINEZ-JULVEZ • Institute of Biocomputation and Physics of Complex Systems
(BIFI), Universidad de Zaragoza, Zaragoza, Spain; Departamento de Bioquı́mica y
Biologı́a Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain
MILAGROS MEDINA • Institute of Biocomputation and Physics of Complex Systems (BIFI),
Universidad de Zaragoza, Zaragoza, Spain; Departamento de Bioquı́mica y Biologı́a
Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain; Fundación
ARAID, Diputación General de Aragón, Aragón, Spain
DENGMING MING • Department of Physiology and Biophysics, School of Life Science,
Fudan University, Shanghai, China
 Contributors xvii

AMIE D. MOODY • Department of Pharmaceutical Sciences, University of Colorado,

Aurora, CO, USA
JAMES M. MOTTONEN • Department of Physics and Optical Science, University of North
Carolina at Charlotte, Charlotte, NC, USA
OWEN W. NADEAU • Department of Biochemistry and Molecular Biology,
University of Kansas Medical Center, Kansas City, KS, USA
CHARULATA B. PRASANNAN • Department of Biochemistry and Molecular Biology,
The University of Kansas Medical Center, Kansas City, KS, USA
ANITA M. PREININGER • Department of Pharmacology, Vanderbilt University
Medical Center, Nashville, TN, USA
GREGORY D. REINHART • Department of Biochemistry and Biophysics, Texas A&M
University and Texas AgriLife Research, College Station, TX, USA
JAMES P. ROBBLEE • Department of Pharmaceutical Sciences, University of Colorado,
Aurora, CO, USA
MARTIN K. SAFO • Department of Medicinal Chemistry, School of Pharmacy & Institute
for Structural Biology and Drug Discovery, Virginia Commonwealth University,
Richmond, VA, USA
JAI A. P. SHANATA • Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA, USA
RYAN P. STAFFORD • Department of Chemistry, The University of Alabama
at Birmingham, Birmingham, AL, USA
DEIRDRE STEWART • Department of Anesthesia Critical Care & Pain Medicine,
Massachusetts General Hospital, Boston, MA, USA
QINGLING TANG • Department of Biochemistry and Molecular Biology, The University
of Kansas Medical Center, Kansas City, KS, USA
TARJANI M. THAKER • Department of Biochemistry, Vanderbilt University Medical
Center, Nashville, TN, USA
CUIJUAN TIE • Department of Biochemistry and Biophysics, Texas A&M University
and Texas AgriLife Research, College Station, TX, USA
ASHUTOSH TRIPATHI • Department of Medicinal Chemistry, School of Pharmacy &
Institute for Structural Biology and Drug Discovery, Virginia Commonwealth
University, Richmond, VA, USA
SONIA VEGA • Institute of Biocomputation and Physics of Complex Systems (BIFI),
Universidad de Zaragoza, Zaragoza, Spain; Departamento de Bioquı́mica y Biologı́a
Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain; Fundación
ARAID, Diputación General de Aragón, Aragón, Spain
ADRIAN VELAZQUEZ-CAMPOY • Institute of Biocomputation and Physics of Complex
Systems (BIFI), Universidad de Zaragoza, Zaragoza, Spain; Departamento de
Bioquı́mica y Biologı́a Molecular y Celular, Universidad de Zaragoza, Zaragoza,
Spain; Fundación ARAID, Diputación General de Aragón, Aragón, Spain
DEEPTAK VERMA • Department of Bioinformatics and Genomics, University of North
Carolina at Charlotte, Charlotte, NC, USA
P. KEITH VERONESE • Department of Chemistry, The University of Alabama
at Birmingham, Birmingham, AL, USA
xviii Contributors

OLEG K. VOROV • Department of Physics and Optical Science, University of North

Carolina at Charlotte, Charlotte, NC, USA
MICHAEL E. WALL • Computer, Computational, and Statistical Sciences Division, Center
for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, NM, USA
LIQUN ZHANG • Department of Physiology and Biophysics, Case Western Reserve
University School of Medicine, Cleveland, OH, USA
 Part I

Monitoring Allosteric Function

 Part II

Monitoring Allosteric Conformational Changes

 Part III

Monitoring Allosteric Changes in Protein

Dynamics/Subpopulation Distribution
 Part IV

Macromolecular and Ligand Engineering

Allosteric Functions
 Part V

Computational Methods/Aids in the Study of Allostery

 INDEX

A Cooperativity ......................................................... 7–9,

14–15, 19, 35, 36, 54, 56, 62–64, 66–68,
Active site........................................................ 92, 222, 71–75, 89, 91, 187, 206, 219, 287, 288,
308, 310–311, 337, 351, 352, 356, 359, 299, 376
361, 370, 376, 381, 424 Correlated mutation...................................... 385–395
Adenosine 5’-diphosphate (ADP) ...................... 8–12, Coupled harmonic oscillators ............................91, 92
72, 73, 88, 117, 312–313, 339, 342, 356, Coupling 31–50, 54, 68, 71–94, 97–112, 121, 187,
370–372, 374, 377–378, 401, 413, 417 235–236, 238, 246, 251, 254, 255,
Allosteric .......................................... 3, 31, 53, 71, 97, 280, 287, 288, 299, 300, 336, 343–346,
133, 175, 187, 206, 217, 235, 261, 279, 307, 385, 386, 390, 391
335, 351, 369, 385, 399, 423 Covariance ...........................................251, 386–393,
coupling free energy.................... 32–34, 275–276 407
free energy ........... 71, 76, 89, 262, 298–299, 392 Cross-correlation analysis.............................. 251, 254
inhibitor .................... 68, 90, 129, 225, 227–229,
353, 370, 373, 374, 379–381 D
interactions ...............................3, 7–8, 54, 55, 67,
68, 73, 85, 90, 94, 124, 129, 176, 187, Diffusion coefficient ..................... 178, 181–182, 185
235–255, 308, 310–312, 336, 352, Dihdrofolate reductase (DHFR), 8–9, 11, 94
390–392, 399, 419 Distance constraint model (DCM), 280, 292,
model ..................................... 32, 74, 90, 98, 100, 295–296, 298–302
117, 206–207, 217–230, 236, 255, 391, DLS. See Dynamic light scattering
392, 401, 402, 407 Docking .......................354–356, 359–364, 423, 425
regulation hypothesis ................................... 7, 389 DPA. See Dynamics perturbation analysis
switch ............................................... 128–129, 238 Drug screen .......................................... 352, 354, 377
Allostery.................................. 3, 4, 7, 14, 32, 35, 53, Dynamic light scattering (DLS), 175–185
54, 76, 81–82, 90–91, 135–136, 205–215, Dynamics .......................................4, 15, 44, 74, 119,
217–230, 235–255, 261, 298–301, 386, 129, 199, 205, 206, 214, 217, 218, 226,
391–393, 399, 424 235–255, 262, 274, 276, 280, 281, 298,
Amino acids ..........................................50, 94, 97–98, 332, 391, 426
103–104, 108, 111, 120–122, 129–130, 208, Dynamics perturbation analysis (DPA), 423–432
222, 246, 287, 340, 356, 362
Analytical ultracentrifugation (AUC), 176, E
192–198, 202, 220
Assembly ...................................... 20–22, 26, 27, 109, Electron microscopy............................................... 127
110, 145, 175–177, 182, 185, 187–203, Electrophysiology .................................102, 108–110,
217–222, 225, 230, 262, 263, 425 317–318, 320–321, 324–326, 330, 332
AUC. See Analytical ultracentrifugation Etomidate ...................................................... 317–332

C F
Calorimetry..............................10, 37, 38, 44, 46–47, Functional site ....................... 4, 369, 424, 425, 427,
53–68, 77–79, 81, 86, 92, 408, 414–416 429, 430
CD. See Circular dichroism
Chemical crosslinking ......... 118, 121–124, 128, 129 G
Chemical footprinting...........................120–121, 129
Circular dichroism (CD)....... 7–12, 14, 15, 125, 126 GABAA receptor ............................................ 317–332
Coevolution ................................................... 385–395 General anesthesia .................................................. 322
Co-expression ........................................309–312, 318 Global fitting ................................................. 399–419
Conformational change ....................................56–57, Glutamate dehydrogenase (GDH)..................... 9–12,
87–88, 104, 117–121, 124–128, 133, 136, 72, 73, 84, 85, 87
137, 169, 206, 217, 239–240, 246, 255, G protein coupled receptors (GPCRs)......... 133–172
279, 353, 399 GTPase-effector interaction.......................... 236, 238

Aron W. Fenton (ed.), Allostery: Methods and Protocols, Methods in Molecular Biology, vol. 796,
DOI 10.1007/978-1-61779-334-9, # Springer Science+Business Media, LLC 2012

437
438 || A LLOSTERY: METHODS
Index
AND PROTOCOLS

H N
Haptoglobin .......................................... 21–23, 26–28 Network rigidity ............................................ 279–302
Hb. See Hemoglobin Nicotinic acetylcholine receptor (nAChR) . 103–106,
Heat capacity ....................... 10, 12, 56, 68, 75, 287, 108, 110–112
298, 417 Nicotinic receptor .................................................... 97
Hemoglobin (Hb)................................19–29, 54, 85, Nonlinear least squares (NLLS) ...................... 10, 59,
90, 262, 290–291, 352, 358, 386 181–182, 185, 241, 265–267, 327, 329, 400
Hetero-oligomeric protein complexes .................. 119 N-terminus phosphorylation ........................ 335–347
Heterotrimeric G proteins ...................133–136, 142, Nuclear magnetic resonance (NMR)
148–150, 164 relaxation .......................... 236–238, 240–244,
Heterotropic interactions.............. 54–60, 62, 63, 68, 248, 250–251, 253, 255, 352–353
308, 310–313, 315
High-throughput ........................280, 281, 352, 355, O
370–374, 380, 381, 427
Human liver pyruvate kinase (hL-PYK) ...... 335–347, Oxygen binding........................................................ 19
369–381
Hybrid enzymes ..................................................... 308 P
Hydrogen-deuterium exchange...........................118,
261–277 Parameter correlation............................401, 405–407
PDZ domain........................................................... 386
Phosphofructokinase (PFK), 308–312
I Plexin RhoGTPase Binding domain ........... 239, 241,
In silico screening.......................................... 351–364 243–246, 248, 251, 253
Ion channel ................................... 102–104, 106, 392 Polysteric linkage........................................... 175–176
Isothermal calorimetry ........................60, 83–89, 401 Protein-DNA interactions ....................................... 48
Isothermal titration calorimetry (ITC) .................. 35, Protein dynamics235, 236, 241–242, 246, 255, 280,
37, 39, 43, 48, 49, 53–68, 71–73, 77, 81–83, 391, 423, 424, 427, 428
85, 87, 91, 94, 262–263, 353, 413, 415 Protein interactions ...................................39, 47, 91,
235–255, 426
K Protein–protein interaction ................................... 245
Protein structure ....................................7, 12, 44, 92,
Kullback–Leibler divergence.................................. 426 190, 219, 236–237, 243, 255, 281, 282,
285, 288–292, 299, 300, 313, 359, 390,
L 391, 423–425
Proteolysis.............................119–120, 128–130, 273
Ligand binding ..................................................4, 6–8, Pyruvate kinase ............337, 351–364, 370, 391, 401
10, 12, 13, 20–21, 54, 55, 59, 62–67,
71–72, 74–77, 85–87, 91, 262–263, 336, Q
337, 353, 391, 392, 399, 416, 425, 427,
428, 432 Quantitative stability-flexibility relationships
Linkage .......................................... 68, 74–76, 89–91, (QSFR), 298–301
123, 124, 175–176, 187–203, 220, 262, Quaternary structure equilibrium ................ 217–227
263, 270, 353, 401, 408–409
Linked equilibrium................................................335 R
Lipari-Szabo order parameters .............................. 244
Rac1, 239–241, 243–254
M Receptor-mediated nucleotide exchange .... 133, 135,
137, 164, 165
Mass spectrometry.............. 129, 223, 262, 266–270, Reduced b-nicotinamide adenine dinucleotide,
273, 274, 277 NADPH ...................................................60–61
MC analysis. See Monte-Carlo analysis Relative entropy............................................. 425–426
Metalloregulation ........................................ 32–34, 40 Rhodopsin ..................133–139, 141, 142, 144–153,
Metal sensor protein ..........................................31–50 161, 163–165, 167–170, 172
Metals in biology......................................... 33, 36–37 Rnd1, 239–240, 246–254
MI. See Mutual information
Monte-Carlo (MC) analysis ................ 406, 407, 410, S
415, 416, 419
Morpheein ..................................................... 217–230 SAXS. See Small angle X-ray scattering
Mutual information (MI) ........................... 387–389, Sedimentation velocity ........................ 176, 178, 185,
393–395 191–194, 201
 ALLOSTERY: METHODS AND PROTOCOLS | 439
Index |
Signal transduction....................................... 133, 235 Thermodynamics.............................................5–8, 13,
Silent allostery ....................................................71–94 32–38, 43, 54, 55, 58–59, 73, 75–77,
Site-directed mutagenesis ....................101, 104–106, 81, 83, 84, 91, 92, 94, 262, 263,
137, 310, 313, 319, 321–322, 330 280–281, 286–288, 292, 298, 299, 302,
Small angle X-ray scattering (SAXS) ............ 127–128 335, 336, 400
Steroid receptors .................................. 188, 189, 201 Titration calorimetry ................................... 57, 62–64
Stopped-flow ............................................... 22, 26, 27 Transducin .......... 136, 139, 142, 144–145, 153–154
Structure-function study........................................ 280 Two-dimensional (2d) native PAGE ....226, 229–230
Two-state allosteric model............................ 206, 401
T
V
Thermodynamic coupling....... 91, 94, 386, 390, 395
Thermodynamic linkage ............................. 20, 21, 90 Van’t Hoff plots ................................... 77, 79, 80, 89
 Chapter 1

Binding Techniques to Study the Allosteric Energy Cycle

James K. Kranz and José C. Clemente

Abstract
Thermodynamic principles of cooperativity and allostery have long been used as a starting point to begin
understanding the interplay between ligand binding events. Understanding the nature of allosteric effects
requires an experimental technique that can be used to quantify ligand binding energies and simultaneously
give experimental insights into the conformational dynamics at play upon ligand binding. CD spectroscopy
provides macroscopic information about the relative secondary and tertiary structures present in a protein.
Here, we use this spectroscopic technique with thermal shift assays wherein ligand binding constants can be
quantified based on their stabilizing effect against thermally induced protein denaturation. Binding constants
for two ligands are used to determine a pairwise coupling free energy which defines the shared energy that
favors or opposes binding of the second ligand binding event in an allosteric system. In CD-based thermal
shift assays, temperature is the driving force for protein unfolding and can also influence protein conforma-
tional dynamics present in the unbound protein or ligand-bound proteins. Dihydrofolate reductase (DHFR)
and glutamate dehydrogenase (GDH) are proposed as example test systems. NADP and methotrexate bind
DHFR with positive cooperativity. Mammalian GDH exhibits negative cooperativity with respect to binding
of NAD and NADPH coenzyme molecules, activation by ADP, and inhibition by GTP.

Key words: Ligand binding, Allosteric energy cycle, Allostery, Thermal shift, Circular dichroism

1. Introduction

It is challenging to succinctly summarize the term “allostery”

given its more than 60-year history applied to innumerable sys-
tems, not only because of the large volume of publications on the
subject but also due to the system-specific nature of allosteric
interactions. As Gregorio Weber pointed out in 1972 (1):
In considering the possible classes of chemical equilibria in proteins in
solution, we are naturally led to distinguish the following possibilities:
(a) the binding equilibria between the protein and added ligands, usually
small molecules; (b) the internal, first-order equilibria between parts of
the same peptide chain; (c) the second- and higher order equilibria
among the several covalent chains that constitute the multi-chain pro-
teins, or among the macromolecules in single-chain proteins. Evidently
no one of these equilibria can be treated in isolation from the others.

Aron W. Fenton (ed.), Allostery: Methods and Protocols, Methods in Molecular Biology, vol. 796,
DOI 10.1007/978-1-61779-334-9_1, # Springer Science+Business Media, LLC 2012

3
4 J.K. Kranz and J.C. Clemente

We now have a wealth of experimental data indicating proteins

naturally exist in a constant state of motion, described by popula-
tions of conformations. This is in fact the source of ligand-induced
protein folding, or local folding coupled to binding, and can be
the source of great specificity (2–5) or enormous plasticity (6–8)
in ligand recognition. Motions coupled to enzyme catalysis have
been long hypothesized as the source of enormous rate accelera-
tions relative to their solution counterparts; the change in dynamics
of a functioning enzyme has recently comes to light (9, 10).
Ligands can shift the population of protein conformational states
in unpredictable ways; in general, binding induces rigidity in a
complex manner (8, 11, 12), but at least one example of an
enzyme shows an increase in conformational heterogeneity upon
substrate binding wherein key conformers become populated only
under steady-state conditions (13).
An allosterically modulated system is one in which the native,
unliganded state of the protein has multiple conformations that
are accessible and that ligand binding causes a shift in the popula-
tion of states owing to an energetic preference for the ligand to
bind to a particular structural form of the protein. In other words,
the allostery arises from the interdependence of ligand binding
events that modulates protein conformational heterogeneity.
Likewise, allostery in enzymatic function arises from the interde-
pendence of substrate binding, catalysis, and product release steps
each with their own unique relationship to the population of
conformational states available to the protein.
The term “allostery” typically is associated with the complex
functions of a multidomain protein, where the fractional satura-
tion by endogenous ligands is characterized by site-resolved bind-
ing or kinetic constants that are nonequivalent. By definition,
heterotropic allosteric regulation (i.e., allostery) must exhibit
two defining characteristics: (1) binding of the effector ligand to
the macromolecule elicits a change in a functional property of the
macromolecule, either enzyme catalysis or binding of a second
ligand; and (2) the effector binds to the macromolecule at a site
topographically distinct from the functional site. This definition
applies to monomeric proteins as well, where the native state is
characterized by conformational dynamic regions that change
upon ligand binding.
Returning again to Weber (1), the concept of allostery lends
itself to an intuitive approach employing the notion of “apparent”
or “conditional” energies. For the simplest case of a monomeric
protein and a single ligand, the protein exists in a number of
conformations, A1. . .AN, that are not equally populated depend-
ing on the energetic stability of each state (14), represented in the
schematic in Fig. 1. Interconversion between any two states, i and
j, is described by a first-order equilibrium constant, k0ij and k1ij,
where the binary subscript 0 denotes a transition in the
 1 Binding Techniques to Study the Allosteric Energy Cycle 5

unliganded state of the protein and 1 denotes the same transition

taking place between liganded forms of the protein. The ligand, X,
has an affinity for every conformation of the protein, given by
Ki(X). The observation of a binding event by a macroscopic
analytical technique yields only an apparent dissociation constant,
Kapp, that encapsulates the interconversion between the unbound
manifold of states and the bond manifold of states:
P 
N
i¼1 ½A i  ½X
Kapp ðXÞ ¼ P  : (1)
N
i¼1 ½A i X

The ensemble of conformations available to the protein in

either the unbound or X-liganded manifolds defined by Kapp(X)
represents the measurable quantity that is the free energy of
binding, DGapp(X) ¼ RT ln Kapp(X). Of course, not all states (or
microstates) are equally probable (15), and the probability of each
state can change upon ligation.
The schematic described in Fig. 1 also extends to a system
containing dual ligands X and Y, which is described by a thermo-
dyanamic cycle (Fig. 2). Equilibrium constants for binding either X
0 0
or Y to an unliganded protein, Kapp (X) or Kapp (Y), respectively, are
equivalent to apparent binding constants in Eq. 1. Equilibrium
dissociation constants for binding the second ligand once a first
1
binding event has occurred are Kapp (X) for binding X to a complex
1
of AY, or Kapp (Y) for binding Y to a complex of AX. We maintain
the formalism that the protein exists in a distribution of states to
emphasize that any binding event can alter the relative population
of states for any part of the thermodynamic cycle.
In any thermodynamic cycle, additivity is required for transi-
tions described as a state function, generally as a free energy of
binding, DG, though enthalpy and entropy, DH and (TDS) are

Fig. 1. A1, Ai, Aj, and AN chain conformations for hypothetical protein. First-order
equilibrium constant of unliganded conformations i and j, k0ij; first-order equilibrium
constant of X-liganded conformations i and j, k1ij. Second-order dissociation constants
of X binding to any individual protein conformation, Ki (X).
6 J.K. Kranz and J.C. Clemente

Fig. 2. Thermodynamic cycle describing a population of states, Ai, for a hypothetical

protein, and binding of either X or Y ligands, or both X and Y. Apparent equilibrium
0 0
constants describe binding of the first ligand, Kapp (X) or Kapp (Y), or binding of the
1 1
second ligand to a complex of the protein and the other ligand, Kapp (X) or Kapp (Y).

also state functions and thus can be used to further dissect DG in a

similar manner. The overall free energies of the system are described
by DGapp(X), DGapp(Y), and DGapp(XY), where both ligands bind
simultaneously, and at topographically nonoverlapping sites. From
Fig. 2, free energy conservation requires that:
DGapp ðXÞ ¼ RT ln Kapp
0
ðXÞ ¼ DGapp
0
ðXÞ; (2)

DGapp ðY Þ ¼ RT ln Kapp
0
ðY Þ ¼ DGapp
0
ðY Þ; (3)

DGapp ðXYÞ ¼ RT ln Kapp

0
ðXÞ þ RT ln Kapp
1
ðYÞ
¼ DGapp
0
ðXÞ þ DGapp
1
ðYÞ; (4a)

¼ RT ln Kapp
0
ðYÞ þ RT ln Kapp
1
ðXÞ
(4b)
¼ DGapp
0
ðYÞ þ DGapp
1
ðXÞ;

¼ DGapp
0
ðXÞ þ DGapp
0
ðYÞ þ DGc ; (4c)

DGc ¼ DGapp
1
ðXÞ  DGapp
0
ðXÞ; (5a)

¼ DGapp
1
ðYÞ  DGapp
0
ðYÞ: (5b)
Free energy terms in Eqs. 2–4c are only “apparent” in the sense
that they encompass ligand binding to a distribution of protein
conformational states that are not defined. They are precisely the
DG values one measures experimentally as the binding energy of
X, Y or both X and Y. Moreover, the thermodynamic cycle is additive,
but not necessarily symmetric, as defined by a coupling free energy
term DGc (1, 16–18). As shown in Eq. 5a, 5b, DGc is measured
experimentally from either DGapp0
(X)  DGapp0
(X), or from DGapp1
 1 Binding Techniques to Study the Allosteric Energy Cycle 7

(Y) DGapp0
(Y). Positive cooperativity is observed when DGapp
1
(X)
< DGapp (X) or when DGapp (Y) < DGapp (Y).
0 1 0

Practically speaking, cooperativity is a thermodynamic

description of interdependent binding events. Heterotrophic allo-
stery further describes the relationship between multiple ligand
binding events that modulate the affinity of each other via confor-
mational changes; i.e., binding results in a change of the relative
populations among possible protein conformational states.
Understanding the underlying mechanisms that are described by
cooperativity and allostery requires a deep investigation using
multiple approaches, thus beyond the scope of any one technique.
What is perhaps most important from a practical sense is defining:
(1) that the system of interest has two different ligands that are
functionally relevant; (2) they are either competitive or bind non-
competitively, in topographically distinct locations; and (3) an
experimental method that provides some insights as to whether
or not allostery exists in the system of interest.
Circular dichroism (CD) spectroscopy has been used exten-
sively to characterize protein folding and unfolding, both as a
function of denaturants and temperature. It is a truly general
technique that is not limited to constraints of molecular size or
chemical composition, is a “label-free” approach that does not
require secondary conjugation or chemical modification to obtain
data on protein structural composition. CD has unique resolving
power in estimating the relative fraction of secondary structural
elements of a protein, as described extensively by Greenfield
(19–23) as well as many others, too numerous to elaborate.
Moreover, modern improvements in CD instrumentation and
data fitting makes it straightforward to collect full spectra as a
function of temperature, providing a means to quantitate the
presence of subtle fluctuations in protein structure that occurs at
temperatures below the primary protein unfolding transition.
What is lacking in the literature, however, is the use of CD
to measure ligand binding; in particular, the combination of CD
as a tool to monitor ligand-induced thermal shifts. Experimen-
tally, replicate protein unfolding experiments are performed at
different concentrations of ligands; the Tm value representing
the mid-point unfolding temperature is related to the total ligand
concentration, from which a KD is obtained. When coupled with
additional information on the composition of the ligands and
protein, binding affinities from thermal shift analyses can be cou-
pled to insights on the changes of protein structure in response to
temperature or ligand binding, supporting a hypothesis of struc-
tural changes linked to binding in reference to model systems.
Thus, for a system that is thought to be allosteric, CD provides a
useful first tool to fully characterize binding of both ligands
independently, to establish the presence of cooperative binding
8 J.K. Kranz and J.C. Clemente

when both ligands are present via thermodynamic cycles, and to

provide a first approximation of allosteric interactions that can be
further elaborated by other techniques.

2. Materials

1. Test protein of interest, such as bovine liver dihydrofolate

reductases, bDHFR (Sigma-Aldrich D6385), or bovine glu-
tamate dehydrogenase, bGDH (Sigma-Aldrich G7882) in a
suitable assay buffer.
2. Ligands of interest, such as NADPH (Sigma: N9660, or
equivalent), methotrexate (Sigma: M9929, or equivalent),
ADP (Fluka 01897, or equivalent), GTP (Fluka 01897, or
equivalent).
3. CD spectrophotometer, such as the Olis DSM CD or equiva-
lent, outfitted with a Peltier-based thermal cell holder and
appropriate quartz cuvettes.

3. Methods

The essentials of CD spectroscopy have been exhaustively elabo-

rated in the literature (see Greenfield (20–27) and references
therein) and will not be detailed here. Historically, measurements
of CD signal at single wavelengths have been employed to follow
the kinetics and thermodynamics of protein folding, the major
advantage being rapid data collection and simplified data analysis.
The limitation of this approach is the loss of information content
that is present in monitoring unfolding over a wide range of
wavelengths. Modern improvements in both instrumentation
and data analysis allow researchers to employ methods such as
singular value decomposition (SVD) (28) or similar methods
(20), toward the interpretation of CD spectra. The advantage of
full spectral analyses applied to allosteric binding is the ability to
uniquely quantify the fractional change in a-helix, b-sheet, and
random coil upon ligand binding. Here, we encourage the use of
full CD spectra in monitoring thermal shift assays for the purpose of
gaining some insights into protein conformational changes that
may be coupled to binding, though a single wavelength is sufficient
for KD determination in a CD-based thermal shift assay (Table 1).
DHFR represents a suitable test system for exploring positive
cooperativity via CD-based thermal shift assays. DHFR is a well-
characterized system with ligand-induced conformational changes
 1 Binding Techniques to Study the Allosteric Energy Cycle 9

Table 1
Features of protein CD spectra

Far-UV CD
Range (190–240 nm)
Source of signal Peptide bond in asymmetric environment;
protein secondary structure
a-Helix Negative at 208 nm
Negative at 222 nm
Positive at 192 nm
b-Sheet Negative at 218 nm
Positive at 196 nm
Random coil Positive at 212 nm
Negative at 195 nm
Near-UV CD
Range (260–320 nm)
Source of signal Aromatics in asymmetric environment;
protein tertiary structure

and positive cooperativity in binding of NADPH and the anti-

folate compound methotrexate (29). Likewise, GDH represents
a suitable test system for exploring negative cooperativity via
CD-based thermal shift assays. GDH is allosterically regulated
by GTP, ADP, and coenzymes NADH or NADPH (30, 31), and
is known to change protein conformation in a ligand-dependent
manner (32). Though DHFR and GDH are presented as
specific examples for sample preparation and data collection,
experiments will be discussed generally without specific analysis
of either system.

3.1. Sample 1. Compound solutions: Test ligands are prepared in DMSO (or
Preparation and Data aqueous buffer if solubility is sufficiently high) at a 50- to 100-
Collection fold concentrated solution, generally in the 10–100 mM
range. Compounds may be diluted in DMSO serially, then
added to protein in a uniform dilution. A typical thermal shift
assay employs 8–12 different concentrations of a test com-
pound with a single negative control (DMSO alone), span-
ning a final concentration range from 0.05 to 200 mM, for
example.
2. Protein solution: The protein is diluted from a concentrated
stock to a working concentration of ~0.5–20 mM protein into
a suitable assay buffer; sufficient volume should be prepared
10 J.K. Kranz and J.C. Clemente

for a unique CD sample at each test concentration, then

aliquoted into microcentrifuge tubes. The exact concentra-
tions of protein and dye are defined by experimental assay
development studies.
3. Compound addition/equilibration: compounds are diluted
uniformly by a factor of 50- to 100-fold into the aliquoted
protein, mixing via pipettor or gentle vortexing.
4. Individual samples are placed into CD cuvettes, and thermally
denatured. Either a single wavelength or spectra are collected
as a function of temperature (see Notes 1–3).

3.2. Thermal Shift The details of thermal shift analysis for ligand binding have been
Assay Data Analysis described for calorimetric studies (33, 34) and for fluorescent-
based probe studies (35–37), and the thermodynamic analysis also
applies to CD experiments. To measure the strength of ligand
binding from a thermal shift assay, the data are fit in two stages.
These are described by Eqs. 6 and 7a, 7b.
yðT Þ ¼ yF;Tm þ mF ðT  Tm Þ
yU;Tm  yF;Tm þ mU ðT  Tm Þ
þ : (6)
DHU;Tr þDCp;U ðT Tr ÞT ðDSU;Tr þDCp;U lnðT =Tr ÞÞ=RT
1þe
The temperature-dependent CD signal, y(T ), is used to deter-
mine the protein thermal stability, Tm, at any given concentration of
ligand (see Note 4). The protein folding free energy, DGU(T ) must
be replaced by the Gibbs–Helmholtz relationships (38), and is com-
posed of a temperature-dependent enthalpy, DHU(T ), and a tem-
perature-dependent entropy, DSU(T ); a heat capacity for protein
unfolding, DCp,U, defines the temperature dependence of DHU,Tr
and DSU,Tr. Temperature-dependent baselines for fully folded
and fully unfolded protein are described by linear functions:
yF ðT Þ ¼ yF;Tm þ mF ðT  Tm Þ and yU ðT Þ ¼ yU;Tm þ mU ðT  Tm Þ,
for folded and unfolded, respectively. The assumption of linear
baselines may not be valid when protein fluctuations occur as a
function of temperature.
Figure 3a shows example protein unfolding data for human
GDH alone and in the presence of ADP. Protein melting was
monitored by CD at 220 nm, fit to Eq. 6 by nonlinear least
squares minimization algorithm to estimate the parameters of
yF;Tm and mF, yU;Tm and mU, DHU;Tr , DSU(T ), and Tm for each
individual sample. DCp,U is always highly correlated with DHU;Tr
in parameter estimation, and is thus difficult to determine inde-
pendently from DHU;Tr with nonlinear least squares regression
analysis. The value of DCp,U was held fixed near its value measured
by other techniques, generally estimated from differential scan-
ning calorimetry (DSC), or is estimated based on protein
composition (39).
 1 Binding Techniques to Study the Allosteric Energy Cycle 11

Fig. 3. (a) Thermally induced unfolding of (10 mM) bovine glutamate dehydrogenase
(Sigma-Aldrich), monitored at 220 nm. Unfolding was performed on the apo protein
(squares) or in the presence of either 250 mM ADP (circles) or 1 mM ADP (triangles).
Data are fit to Eq. 7a, 7b, which gave Tm ¼ 54.2 (0.1) C, DHapp ¼ 90 (15) kcal/
mol for apo DHFR; baseline parameters were yf ¼ 227,000 (deg cm2 mol1) and
mf ¼ 620 (deg cm2 mol1 C1) for the native state, and yu ¼ 65,400
(deg cm2 mol1) and mu ¼ 980 (deg cm2 mol1 C1) for the denatured state. Similar
baseline values were obtained for ADP-bound transitions. (b) Plots of Tm versus ligand
concentration, used to fit a binding constant of ADP to GDH.

Figure 3a also shows thermal unfolding of GDH in the

presence of either 250 mM ADP or 1 mM ADP. As expected,
GDH is stabilized against unfolding with an elevation of Tm in
proportion to increasing [ADP]. What is more interesting is the
behavior of GDH protein conformation in the pretransition
region. At temperatures below ~30 C, both the apo and ADP-
bound forms (250 mM and 1 mM) of GDH show common
baseline behavior, with similar slopes and similar CD spectra
(data not shown). However, in the 35–50 C range, the apo
12 J.K. Kranz and J.C. Clemente

form deviates from linear behavior of ADP-bound GDH, with a

change in elipticity at 220 nm consistent with an increase in
random coil character. This observation is suggestive of a temper-
ature-dependent change in the distribution of GDH conforma-
tional states in the 35–50 C range, specific to the unliganded
form. Binding of ADP induces a change in protein structure that
is consistent with the CD characteristics of the apo state near room
temperature. Here, we can see the limitations of using single
wavelength CD data in studying protein conformations in CD-
based thermal melts. Further evaluation using CD spectral ana-
lyses of temperature-dependent conformational changes (see
Greenfield (20–27)) and references therein) would be needed to
ascertain a connection to allosteric ligand binding.

3.3. Binding In a second stage of thermal shift data analysis, the Tm value of the
Constants from protein at each concentration of ligand is related to the expected
Thermal Shift Assays effect for a ligand with a given binding affinity, Kb or KD. The
relationship between total ligand concentration, total protein
concentration, and the two equilibrium constants for protein
unfolding and ligand binding is defined by Eq. 7a, in terms of
equilibrium constants; data must be fit using Eq. 7b which
expresses equilibrium constants in terms of their temperature-
dependent forms:
 
Pt 1
Lt ¼ ð1  KU Þ þ ; (7a)
2 KU Kb
 
Lt ¼ 1  eðDHU;Tr þDCp;U ðTm Tr ÞTm ðDSU;Tr þDCp;U lnðTm =Tr ÞÞÞ=RTm

Pt 1
 þ (7b)
2 e ð DH U;Tr þDC p;U ðTm T r ÞTm ðDSU;Tr þDCp;U lnðTm =Tr ÞÞÞ=RTm

 eðDHb ðT0 ÞþDCp;b ðT T0 ÞT ðDSb ðT0 ÞþDCp;b lnðT =T0 ÞÞÞ=RT

Pt is the total protein concentration (sum of U, N, and NLb for

unfolded, native, or ligand-bond forms). Lt is the total ligand
concentration (sum of Lf and NLb for free or protein-bound
forms). KU is the protein unfolding equilibrium constant in
both Eqs. 6 and 7a, 7b; Kb or KD is described in terms of an
enthalpy, DHb(T0), an entropy, DSb(T0), and heat capacity, DCp,b,
of ligand binding in Eq. 7b.
Figure 3b shows an example of binding constant determination
for ADP binding to GDH. Protein thermal melting temperatures,
Tm, are plotted as a function of ligand concentration, and fit
to Eq. 7a, 7b. ADP stabilized the protein against unfolding:
Tm ¼ 63.4 C with 250 mM ADP, Tm ¼ 66.0 C with 650 mM
ADP, and Tm ¼ 69.4 C with 1 mM ADP for the three data points
shown as an example. Binding constants are estimated at an arbitrary
 1 Binding Techniques to Study the Allosteric Energy Cycle 13

reference temperature, generally at 25 or 37 C; here, KD(37 C)

¼ 3 mM and DG(37 C) ¼ 7.8 kcal/mol for a binding free energy.
Each ligand should be assayed for binding by thermal shift
assays independently, to determine DGapp 0
(X) and DGapp
0
(Y). Gen-
erally, protein thermal denaturation should be performed at several
different concentrations of ligand (n > 6, preferably n > 10) for
each KD measurement. Theoretical curves are generated to dem-
onstrate how the dependence of Tm on ligand concentration is
affected by several important parameters in the thermal shift assay.
The theoretical behavior of weak to tight binding ligands based on
variations in theoretical affinity is shown (Fig. 4a). The KD greatly

Fig. 4. Simulations of thermal shift concentration response curves using Eq. 7a, 7b. The
thermodynamic parameters describing protein stability were held constant for all simula-
tions. (a) Effect of differing ligand binding affinities ranging from KD ¼ 0.001–10 mM, with
a constant protein concentration, Pt ¼ 1 mM. (b) Effect of varying protein concentrations
from Pt ¼ 0.0001–10 mM, with a constant ligand binding affinity, KD ¼ 0.001 mM.
14 J.K. Kranz and J.C. Clemente

affects the magnitude of DTm at a given concentration of added

ligand. For the two weakest simulated binding curves, there is no
change in Tm at ligand concentrations below their respective ligand
affinities. Simulated curves for tight binding inhibitors highlight
the influence of the protein concentration on the shape of the
dose–response curve. In these simulations, a sigmoidal dependence
of Tm on ligand concentration is observed just below the protein
concentration, consistent with a saturation-binding experiment.
This aspect of thermal shift assays is a unique consequence of the
Gibbs–Helmholtz equation (33, 34), the result of which is a
continuing shift in the equilibrium between native and denatured
protein as more and more ligand is added.
Simulations in Fig. 4b further explore the affect of protein
concentration on the shape of the dose–response curve, assuming
a constant high affinity of KD ¼ 1 nM. Simulations varied the
protein concentration from [Pt] ¼ 0.1, 1, or 10 mM as well as the
theoretical limit where the protein concentration is infinitely
dilute ([Pt] << KD). In each of the examples where the protein
concentration exceeds the KD, a sigmoidal dependence of Tm on
ligand concentration is observed until protein saturation is
achieved. This phenomenon also gives insight into the binding
stoichiometry between ligand and protein.
Following determination of KD values for X and Y ligands
individually, a concentration of either X or Y is selected for
subsequent cooperativity studies; at tenfold above the KD, the
protein should be sufficiently saturated with either X or Y to test
cooperative binding of the alternative ligand, though iterative
testing at different concentrations may be required. Samples are
prepared again, including a common constant concentration of the
secondary ligand. For example, in testing binding of X at different
concentrations, all samples contain Y at a fixed concentration. Note
that the coupling free energy can be determined from either com-
0 0 1
bination of Kapp (X) and Kapp (Y) and Kapp (Y); it is not necessary to
determine all four (though it is recommended to confirm additivity
around the thermodynamic cycle). All binding constants are con-
verted to a free energy of binding, and the coupling free energy is
determined.
Spectral analysis of the CD data should be performed to
ascertain fluctuations in the native state upon binding either X
or Y, and importantly with both ligands bound. In addition,
the temperature dependence of native state structure and of
ligand-bound states can provide additional evidence of changes
in protein conformational states. In order to interpret a CD
change as a change in the dynamics of the protein, full spectral
analysis is strongly encouraged. Finally, we wish to emphasize the
use of this method as a potential litmus test for the presence of
 1 Binding Techniques to Study the Allosteric Energy Cycle 15

cooperativity and possible allostery; the data should inspire fol-

low-up experimentation that is better suited for characterizing the
precise mechanism of allosteric binding.

4. Notes

1. No two CD spectrophotometers have the same dynamic

range and operational restrictions; consult the instrument
manual or manufacturer for specifics.
2. For reference, typical wavelengths used in CD spectroscopy
are shown in Table 1. For a protein with significant a-helical
structure, unfolding may be monitored at 222 and 208 nm.
Recommended wavelength ranges for analyzing secondary
structure vary (20), but typically encompass 200–240 nm.
3. For thermal shift assays, the experimental temperature is
steadily increased at such a rate as to allow thermal equilib-
rium to be maintained throughout the experiment. Typical
temperature ramp rates range from 0.1 to 10 C/min (com-
monly 1 C/min). The spectra or individual wavelength data
are collected at regular intervals, 0.2–1 C/image, over a tem-
perature range spanning the typical protein unfolding tem-
peratures of 25–95 C.
4. Equations in Subheading 3.2 used for fitting protein thermal
stability values, Tm, assume a linear dependence on tempera-
ture. If there is a temperature-dependent change in protein
conformation or distribution of conformations, the native
state baseline may not adhere to a linear temperature depen-
dence. A rigorous analysis of CD spectra as a function of
temperature, as suggested above (but is beyond the scope of
the current paper), is necessary to lend interpretation to tem-
perature-dependent structural changes.

References
1. Weber, G. (1972) Ligand binding and internal N-terminal RNA-binding domain of human
equilibria in proteins. Biochemistry 11, U1A protein. J Mol Biol 322, 533–54
864–878 5. Shoemaker, B. A., Portman, J. J., and
2. Kranz, J. K., and Hall, K. B. (1999) RNA Wolynes, P. G. (2000) Speeding molecular
recognition by the human U1A protein is recognition by using the folding funnel: the
mediated by a network of local cooperative fly-casting mechanism. Proc Natl Acad Sci
interactions that create the optimal binding U S A 97, 8868–887
surface. J Mol Biol 285, 215–231 6. Lee, A. L., and Wand, A. J. (2001) Micro-
3. Showalter, S. A., and Hall, K. B. (2004) Alter- scopic origins of entropy, heat capacity and
ing the RNA-binding mode of the U1A RBD1 the glass transition in proteins. Nature 411,
protein. J Mol Biol 335, 465–480 501–504
4. Showalter, S. A., and Hall, K. B. (2002) 5. 7. Brokx, R. D., Lopez, M. M., Vogel, H. J., and
A functional role for correlated motion in the Makhatadze, G. I. (2001) Energetics of target
16 J.K. Kranz and J.C. Clemente

peptide binding by calmodulin reveals differ- 22. Greenfield, N. J. (2006) Analysis of the kinetics
ent modes of binding. J Biol Chem 276, of folding of proteins and peptides using circular
14083–14091 dichroism. Nat. Protocols 1, 2891–2899
8. Frederick, K. K., Marlow, M. S., Valentine, K. 23. Greenfield, N. J. (2006) Using circular
G., and Wand, A. J. (2007) Conformational dichroism collected as a function of tempera-
entropy in molecular recognition by proteins. ture to determine the thermodynamics of pro-
Nature 448, 325–329 tein unfolding and binding interactions. Nat.
9. Henzler-Wildman, K., and Kern, D. (2007) Protocols 1, 2527–2535
Dynamic personalities of proteins. Nature 24. Adler, A. J., Greenfield, N. J., and Fasman,
450, 964–972 G. D. (1973) Circular dichroism and optical
10. Nashine, V. C., Hammes-Schiffer, S., and rotatory dispersion of proteins and polypep-
Benkovic, S. J. (2010) Coupled motions in tides. Methods Enzymol. 27, 675–735
enzyme catalysis. Curr Opin Chem Biol 14, 25. Greenfield, N. J. (2004) Circular dichroism
644–651 analysis for protein-protein interactions. Meth-
11. Tsai, C. J., del Sol, A., and Nussinov, R. ods Mol. Biol. 261, 55–78
(2008) Allostery: absence of a change in 26. Greenfield, N. J. (2004) Analysis of circular
shape does not imply that allostery is not at dichroism data. Methods Enzymol. 383,
play. J Mol Biol 378, 1–11 282–317
12. Wand, A. J. (2001) Dynamic activation of pro- 27. Greenfield, N. J. (2006) Determination of the
tein function: a view emerging from NMR folding of proteins as a function of denatur-
spectroscopy. Nat Struct Biol 8, 926–931 ants, osmolytes or ligands using circular
13. Larion, M., Salinas, R. K., Bruschweiler-Li, L., dichroism. Nat. Protocols 1, 2733–2741
Bruschweiler, R., and Miller, B. G. (2010) 28. Hennessey, J. P., Jr., and Johnson, W. C., Jr.
Direct evidence of conformational heteroge- (1981) Information content in the circular
neity in human pancreatic glucokinase from dichroism of proteins. Biochemistry 20,
high-resolution nuclear magnetic resonance. 1085–1094
Biochemistry 49, 7969–7971 29. Bystroff, C., and Kraut, J. (1991) Crystal
14. Hilser, V. J., Garcia-Moreno, E. B., Oas, T. G., structure of unliganded Escherichia coli
Kapp, G., and Whitten, S. T. (2006) A statisti- dihydrofolate reductase. Ligand-induced con-
cal thermodynamic model of the protein formational changes and cooperativity in bind-
ensemble. Chem Rev 106, 1545–1558 ing. Biochemistry 30, 2227–2239
15. Vertrees, J., Wrabl, J. O., and Hilser, V. J. 30. Frieden, C. (1965) Glutamate dehydrogenase.
(2009) Energetic profiling of protein folds. VI. survey of Purine Nucleotide and other
Methods Enzymol 455, 299–327 effects on the enzyme from various sources.
16. Di Cera, E. (1998) Site-specific analysis of J Biol Chem 240, 2028–2035
mutational effects in proteins. Adv Protein 31. George, A., and Bell, J. E. (1980) Effects of
Chem 51, 59–119 adenosine 50 -diphosphate on bovine gluta-
17. Jencks, W. P. (1981) On the attribution and mate dehydrogenase: diethyl pyrocarbonate
additivity of binding energies. Proc Natl Acad modification. Biochemistry 19, 6057–6061
Sci U S A 78, 4046–4050 32. Banerjee, S., Schmidt, T., Fang, J., Stanley, C.
18. Weber, G. (1975) Energetics of ligand binding A., and Smith, T. J. (2003) Structural studies
to proteins. Adv Protein Chem 29, 1–83 on ADP activation of mammalian glutamate
19. Greenfield, N., and Fasman, G. D. (1969) dehydrogenase and the evolution of regula-
Computed circular dichroism spectra for the tion. Biochemistry 42, 3446–3456
evaluation of protein conformation. Biochem- 33. Brandts, J. F., and Lin, L. N. (1990) Study of
istry 8, 4108–4116 strong to ultratight protein interactions using
20. Greenfield, N. J. (1996) Methods to estimate differential scanning calorimetry. Biochemistry
the conformation of proteins and polypeptides 29, 6927–6940
from circular dichroism data. Anal Biochem 34. Shrake, A., and Ross, P. D. (1990) Ligand-
235, 1–10 induced biphasic protein denaturation. J Biol
21. Greenfield, N. J. (2006) Determination of the Chem 265, 5055–5059
folding of proteins as a function of denatur- 35. Kranz, J. K., and Schalk-Hihi, C. (2011) Protein
ants, osmolytes or ligands using circular thermal shifts to identify low molecular weight
dichroism. Nat. Protocols 1, 2733–2741 fragments. Methods Enzymol 493, 277–98
 1 Binding Techniques to Study the Allosteric Energy Cycle 17

36. Matulis, D., Kranz, J. K., Salemme, F. R., and 38. Privalov, P. L. (1979) Stability of proteins:
Todd, M. J. (2005) Thermodynamic stability small globular proteins. Adv Protein Chem
of carbonic anhydrase: measurements of 33, 167–241
binding affinity and stoichiometry using Ther- 39. Murphy, K. P., and Gill, S. J. (1991) Solid
moFluor. Biochemistry 44, 5258–5266 model compounds and the thermodynamics
37. Zhang, R., and Monsma, F. (2010) Fluores- of protein unfolding. J Mol Biol 222,
cence-based thermal shift assays. Curr Opin 699–709
Drug Discov Devel 13, 389–402
 Chapter 2

Kinetic Trapping of a Key Hemoglobin Intermediate

Jo M. Holt and Gary K. Ackers

Abstract
The complete binding cascade of human hemoglobin consists of a series of partially ligated intermediates.
The individual intermediate binding constants cannot be distinguished in O2 binding curves, however,
each constant can be determined from the O2-induced change in assembly constant for the a2b2 tetramer
from its constituent ab dimers. The characterization of these O2 binding constants has shown the Hb
cascade to be asymmetric in nature, with binding dependent upon the specific distribution of O2 among
the four hemesites. A stopped-flow approach to measuring the dissociation constant of a key doubly
ligated intermediate, that in which one dimer is oxygenated and the other is not, is described. The
intermediate is transiently formed in the absence of O2 and then allowed to dissociate in the presence
of O2. The free dimers thus released are trapped by the plasma protein haptoglobin, the rate limiting step
being that of tetramer dissociation. The kinetic constant observed for the dissociation of this intermediate
confirms the value for its equilibrium O2 binding constant, previously determined under equilibrium
conditions by subzero isoelectric focusing.

Key words: Hemoglobin, Cooperativity, Allostery, Thermodynamic linkage, Oxygen binding,

Haptoglobin, Stopped flow

1. Introduction

Human hemoglobin (Hb) is composed of four heme-containing

subunits, two a and two b, each of which binds a single O2 ligand.
The tetramer readily dissociates to its constituent ab dimers,
indicating that the dimer–dimer interface is weaker than the intra-
dimer interface. Interaction among the four subunits is manifested
in a strong positive cooperativity of O2 binding and release. Four-
teen partially ligated intermediates are generated in the course of
O2 binding, each with a different configuration of bound ligand
among the four hemesites (Fig. 1). The binding constant for the
first oxygen ligand, K1, has the same value for all subunits, a1, b1,
a2, and b2. However, binding the second O2 to yield two ligands
on a single ab dimer (tetramer species 21) is approximately tenfold

Aron W. Fenton (ed.), Allostery: Methods and Protocols, Methods in Molecular Biology, vol. 796,
DOI 10.1007/978-1-61779-334-9_2, # Springer Science+Business Media, LLC 2012

19
20 J.M. Holt and G.K. Ackers

species 21
O2 isomers
O2

K2* O2
O2 K3* O2 O2
O2
O2

O2
O2 O 2
O2 O2 O2
α1 β2 K1 K4 O2 O2

β1 α2 O2 O2
O2
K2 K3
O2 O2 O2
O2 O 2

O2 O2 O2
O2
O2
O2

O2

O2

Fig. 1. The Hb binding cascade, showing all possible distributions of bound O2 among the four subunits, including isomeric
forms. The species 21 intermediate is that in which one ab dimer has no ligands and the partner dimer is fully oxygenated.

more favorable than binding the second O2 to yield one ligand on

each dimer (K2* ¼ 10 K2, Fig. 1) (1). This difference demon-
strates that the Hb subunits do not respond uniformly to
oxygenation, contrary to what has historically been accepted (2).
An experimental approach to measuring the intermediate
binding constants for Hb (which are not accessible from analysis
of traditional O2 binding curves) was developed using linkage
thermodynamics, capitalizing on the strict correlation between
the O2 binding constants K1, K2, K2*, K3, K3*, and K4, and the
ab dimer ! a2b2 tetramer assembly constants for each tetramer
(3). Because O2 is an inherently labile Hb ligand, it is necessary to
fix the configuration of bound ligand in the tetramer, and this is
accomplished through the use of nonlabile hemesite analogs, such
as the substitution of Zn(II) heme for the native Fe(II) heme (1).
The intermediate binding constants have been characterized with
a variety of hemesite analogs, in differing solution conditions, and
with both symmetric and asymmetric modifications, providing a
quantitative description of the entire binding cascade (4, 5).
The Zn(II) heme analog does not bind ligands such as O2 and
has proven to be an excellent deoxy hemesite analog (6–8).
Although direct O2 binding to the asymmetric doubly ligated
 2 Kinetic Trapping of a Key Hemoglobin Intermediate 21

species 21 is not experimentally feasible, even with the Zn(II)

heme analog, subzero isoelectric focusing has permitted its dimer
! tetramer equilibrium constant, and thus its O2 binding con-
stant, to be determined via thermodynamic linkage analysis (8, 9).
The difference between the assembly free energy, DG2, of species
21 and a singly ligated species is equal to the difference in free
energy of O2 binding, a value referred to as the cooperative
free energy. When added to the intrinsic (or noncooperative)
free energy of O2 binding (observed with the free ab dimer), the
cooperative free energy yields the O2 binding free energy for any
step in the cascade.
In a kinetic experiment described here, the Zn/FeO2 species
21 tetramer is transiently formed and then allowed to dissociate.
The free dimers thus formed are trapped by the plasma protein
haptoglobin, with the rate limiting step being that of tetramer
dissociation (10). The kinetic constant for dissociation of the Zn/
FeO2 species 21 tetramer is measured in this manner, confirming
the value for the equilibrium O2 binding constant.

2. Materials

2.1. Preparation of 1. Normal saline solution: 0.9% w/v NaCl, i.e., 9 g NaCl in 1 L.
Native FeO2 Tetramers 2. Buffer exchange and concentrators: Sephadex G25 column,
stirred-cells (Amicon), disposable ultrafiltration tubes.
3. Cation exchange chromatography: HPSP Sephadex (Amer-
sham), FPLC (Pharmacia).
4. Chromatography Buffer A: 0.01 M NaH2PO4, 1 mM EDTA,
pH adjusted to 6.8. Store at 4 C.
5. Chromatography Buffer B: 0.02 M Na2HPO4, 1 mM EDTA,
pH adjusted to 8.3. Store at 4 C.
6. Mixed-bed ion exchange resin (BioRad AG501 8).

2.2. Preparation of Zn 1. Acetone (high purity).

Tetramers 2. Concentrated HCl.
3. Ethylene glycol.
4. Methanol.
5. A low-temperature water bath with external circulation, using
tubing that is resistant to methanol and ethylene glycol.
6. Jacketed reaction vessel (2-L), glass only, acid washed.
7. Dialyzate: 1 mM sodium bicarbonate, 0.1 mM dithiothreitol.
8. Zn(II) protoporphyrin IX (Frontier Scientific, Logan, UT).
9. Syringe filters: 0.45 mm cellulose acetate.
22 J.M. Holt and G.K. Ackers

2.3. Preparation of 1. Anaerobic chamber (Coy Laboratory Products, Inc.).

Zn/Fe Asymmetric 2. Deoxygenation procedures were carried out with humidified
Hybrid Species 21u ultra high purity N which was further purified by O -remov-
ing cartridges (QC2 + panel/Agilent Technologies, Supelco),
2
connected to the chamber by stainless steel or copper tubing
(Swagelok).
3. Glass gas-tight syringes (Hamilton).
4. Sodium dithionite. Store in a dessicator in the freezer.
5. Standard buffer: 0.1 M Tris (Trizma base, SigmaUltra), 0.1 M
NaCl (SigmaUltra), 1 mM Na2EDTA (SigmaUltra) titrated at
21.5 C with concentrated HCl at pH 7.4 to give a total [Cl]
of 0.18 M.

2.4. Dimer Trapping 1. Stopped-flow spectrophotometer (Applied Photophysics

with Haptoglobin SX.18MV), customized with an anaerobic chamber (Coy
Laboratory Products, Inc.) enclosing the sample handling
unit of the spectrophotometer.
2. Human haptoglobin type 1:1 (Sigma). Store frozen.

3. Methods

The absorbance decrease in the Soret region that accompanies the

formation of the haptoglobin-deoxy dimer complex has been used
to measure the rate constant of tetramer dissociation, koff, for both
modified and native Hbs over a range of solution conditions
(11–15). In combination with the dimer ! tetramer assembly
rate constant, kon ¼ 1.1  106 M1 s1 (2, 15), the equilibrium
assembly constant can be obtained. The value for kon has been
shown to be invariant among a wide range of modifications and
mutations to the deoxy tetramer (15).
The species 21 tetramer can be formed by hybridizing parent
tetramers ZnHb and native FeO2Hb, however, the maximum
fraction of 21 hybrid formed at equilibrium is only 3% of
the mixture, as measured by subzero isoelectric focusing (8).
However, under anaerobic conditions, the unligated species 21u
tetramer comprises nearly 30% of the hybrid mixture after 24 h of
equilibration (Fig. 2). Addition of O2 to this anaerobic mixture
results in rapid formation of species 21 from 21u, followed
by tetramer dissociation and disproportionation to the parent
species, such that the species 21 fraction of the hybrid
mixture falls from 30 to 3% within a few seconds. The reaction
of haptoglobin with fully ligated species FeO2Hb is rapid (t1/
2 ¼ 1 s), but does not result in an appreciable change in
absorbance. Reaction of haptoglobin with the parent species
Another Random Scribd Document
with Unrelated Content
 Drawn by the cords of love, held by ties too sacred to be
broken, worn by years of poverty and sickness, and moving at last in
a kind of trance, more dead than alive, one of God's truest and
bravest servants blindly stumbled on till she found herself, for a time
of agony, in a spiritual prison-house from which there seemed to be
no escape.
Her greatest danger lay in a kind of fatalistic submission, which
would have meant permanent disloyalty to her own ideals and
convictions, as well as the abandonment of her vocation. She read
the letters of Père Didon, whose heroic acceptance of his destiny
influenced her in the direction of sinking her individuality. And one
cannot understand the exquisite torture of her position unless one
realises that her mind had often been made an arena of conflict
between the apparently irreconcilable claims of the domestic and the
apostolic life. And yet, Father Hyacinth once said to her in Paris, "You
are the only woman I have ever met who has reconciled the
vocation of a mother with that of an apostle." It may be appropriate
to say here that the prayers of both father and mother have been
answered in the conversion of their ten children, who have of their
own free will consecrated themselves to the service of Christ. Four
sons and three daughters are already engaged in active evangelistic
work, and have been used of God for the conversion of many souls.
To bring the Maréchale back to liberty, God used the voices of
nature, of children and of friends.
She had always had a poet's sensitive ear for earth's thousand
voices of praise, and, sitting in a garden on a spring morning, she
wrote: "The past, whoever was right or wrong, shall be buried. Let
the dead bury their dead. Leave it, and this beautiful spring-tide let
us begin again. The crocuses and snowdrops in this lovely garden all
say, 'New Resurrection life!'"
Her own children's voices called her back into the thick of the
old spiritual battle. "Now for the children," she wrote; "they must all
see some salvation work, and they will feel the glow of the heavenly
fire. It will warm them, and say something far more than all the
Bible lessons in the world." Her eldest daughters, at that time girls of
sixteen and fifteen, but with a wisdom far beyond their years,
literally pushed her into the war, and if ever she was unable to go
they buckled on their armour and took her place. "Mother writes
me," says Victoria in her journal, "that Evangeline held a beautiful
meeting on Sunday because she herself was too ill to go. Poor
mother, it is difficult for her to keep up her courage." This diarist of
fifteen thus philosophises on the meaning of her mother's sorrow.
"Everybody can understand why God lets people of the world, the
infidels, the self-seekers, the indifferent, suffer. It is to bring them to
Himself through disappointments in the world, in themselves, and in
others. But why He allows His children that love Him, those whose
greatest wish is to serve Him—why He allows them to suffer is a
mystery. Perhaps it is to bring them into still closer communion with
Himself, so that they may become one with Him—His, body, soul and
spirit, without reserve."
The Maréchale's friends helped to bring her back to her
predestined work of soul-winning.
During a time of awful silence, in which she never received a
call and rarely a letter, she had not courage to visit any of her old
comrades in Paris. Once, in her great sorrow, she wanted to get
away to some sympathising friend and open her heart. At first she
could think of no one, but suddenly she remembered a humble
working woman, and, taking the train to Paris, she wearily climbed
to the fifth story of a house in the Villette, sat down in this woman's
little room, and burst into a flood of tears. Her friend tried to comfort
her and, not quite understanding this passionate grief, made her lie
down in her own bed while she prepared for her a delicious little
French meal.
Twenty years before, on a dark winter night, the Maréchale was
passing along the Seine embankment on the way to her place of
meeting on the Quai de Valmy. She noticed a girl gazing at the dark,
cold waters, and a voice told her that she was meditating suicide.
Touching her arm she said—
"Don't look at those black, cruel waters. Come with me and
have a nice cup of coffee. You seem to be in trouble."
The girl, whose face was dark and sullen, looked at her
suspiciously, and did not speak. The Maréchale gently pleaded with
her to come and hear a lady sing.
"She sings beautifully, and you will find light and warmth and
comfort, and you will have a good cup of coffee. Do come with me."
The girl at length consented and came. She heard the
Maréchale herself sing. She sat right through the service without
opening her lips and with a hard look on her face. At the end the
Maréchale went down beside her, asked if she had enjoyed the
meeting, and said a word to her about the goodness of God. At the
mention of the name of God, the girl burst into passionate speech.
"God! Don't talk to me of God! I hate Him. What has He done
for me? Why did He take my mother? He doesn't care for me. If He
did He would not have let me be born in prison. What have I done
to deserve such a life as this? It isn't my fault."
But, while the Maréchale talked with her and prayed with her,
the girl's heart was softened. She began to attend the meetings, and
soon gave her heart to the Lord Jesus. Born in a prison, and saved
from suicide in the Seine, she became in turn her rescuer's best
comforter in an hour of supreme sorrow.
When the Maréchale at length plucked up heart to revisit
England, after a long absence and silence, her steps were directed
to the home of a dear friend and kindred spirit in South London. It
was in early girlhood that Mrs. Holman of Jerviston was first
attracted to the Maréchale. Her mother had, as Lady Mayoress,
invited Miss Booth, before the work began in France, to address a
drawing-room meeting in her house, and the indelible impressions
made on a receptive mind on that day proved an inspiration to a
lifetime of quiet and devoted service of Christ. But so timid and
dejected had the Maréchale become that she dreaded the reception
that might await her even in the home of a lifelong friend! She
trembled as she dragged herself across Streatham Common. She sat
down on a seat and—her ruling passion strong as ever—spoke to a
beggar about his soul, feeling a certain new kinship with all outcasts
and pariahs. When she stood before her friend's door she scarcely
had courage to ring the bell, and if she had been told to go to the
kitchen and have a cup of tea with the servants, she would have
answered quite simply, "Yes, I will go." But Mr. Holman himself
opened the door, and his hearty welcome and the outflow of a
perfect sympathy at once cast all her fears to the winds. Her friends
were ministering angels to her. They nursed her back to health,
dried her tears, and made her smile. Their little daughter—now one
of the sweetest singers in London—carolled to her every morning
and awoke "the hallelujah bird" again in her own breast. And God
Himself was meanwhile doing for her what even the best of friends
could not do—giving her the Resurrection Life, re-animating her
hope, baptising her afresh with the Spirit, not of fear, but of power
and of love, breaking all shackles and making her free—free from
the ensnaring fear of man, free to obey the Divine call she had
received even as a child—woman's Pentecostal call to prophesy for
Christ, her one and only Master.
Aller An fang ist schwer, and the difficulties which the Maréchale
encountered at the resumption of her work were enough to make
any but the stoutest heart faint. If she had in the past shown a
bravery transcending that of even the bravest all this was of small
account compared to the heroism now required of her. In the past
she had the help of her own people, her spiritual children, and a
strong organization back of her. Now she was utterly alone,
thousands all over the world had an erroneous conception of the
entire situation, and many even believed she had made shipwreck of
her faith. Her daughter Victoria stands out during that lonely period
of beginnings. With remarkably clear judgment, discernment and
sympathy she cheered and inspired her mother with her own
enthusiastic hopefulness and vivid faith.
The greatest service anyone could render the Maréchale was to
bid her go on, fulfil her destiny, believe that God would again
mightily use and bless her. It might seem a small thing to say, "Be of
good cheer," yet it was one of those "little nothings" for which she
was ever afterwards profoundly grateful. I remember her coming
one evening into my Chelsea study in a mood of depression, baffled
by life's insoluble mysteries. Wondering what would lighten for her
the burden and the weary weight, I took down Browning and began
to read "Rabbi ben Ezra," which was new to her. From the opening
words—

The best is yet to be,

The last of life, for which the first was planned—

to the magnificent close, that inspired Sursum Corda thrilled her as a

message direct from the great Heart of God. Only in one thing did
she venture to differ from the poet. "He sees his heaven beyond,"
she said; "I want mine down here in the salvation of souls."
As soon as she resumed her work, she had her reward in signs
following everywhere. Doors opened to her, first in England, then in
Scotland, Ireland and Wales. She brought the breath of life into
many churches, rekindled the zeal of many workers for Christ, and
broke the chains which had bound multitudes of souls to an evil
past.
Her own experience had given her, as a physician of souls,
perhaps a deeper sympathy, a surer insight, a greater power to
grapple with every form of evil than ever she had before. It became
her mission to save people from themselves by convincing them that
only one thing is entirely worth doing—living like Christ by letting
Christ live in them. There are certainly few evangelists who have
changed the whole current of so many lives in our country. Young
ladies about to pass within convent walls have found a more
excellent way by receiving the living Christ into their hearts. Actors
and singers have consecrated their gifts to Christ and His kingdom.
Young men of the world have heard the call of God and resolved to
enter the ministry or go to the mission-field. God's gift of life has
revealed to many questioning eyes its glorious possibilities.
Multitudes who had no faith have heard another say that she has
faith for them—a faith which has somehow dispelled the mists of
doubt and error and brought them into the sunlight of Divine love.
At the same time her ever-deepening knowledge of her two
books, the Bible and the Heart of Man, have made her a unique
preacher to preachers. One night at Keswick, in the summer of 1907,
a brilliant young Scottish minister, who was a member of a large
house-party of clergymen attending the annual Convention, came
home late for supper.
"Excuse me," he said, as he sat down, "but I could not tear
myself away from the open-air meeting in the Square. I never heard
such speaking in my life. I stood transfixed. The preacher was the
daughter of General Booth, and I never knew the English language
was such a magnificent weapon until to-night. Her preaching was
extraordinary."
Next day, through Dr. Harry Guinness, who was her host, she
was invited to address that house-party. Another preacher who was
present has recorded his impressions. "After tea we all gathered our
chairs in a circle round her as she opened up some sacred chapters
in her life. Hour after hour sped. No one thought of moving to go to
the Tent meetings. There we sat spellbound, through a long
evening, feeling we had never come across such a being before. This
was the first of many such meetings, to which as many outsiders
were invited as the large drawing-room could hold. What evenings
these were! Highland worthies sat gazing at her with open-mouthed
wonder, held by her witchery, her strange tales from actual life, by
her wisdom and pathos. Her voice, rich and sweet, sometimes fell to
dreamy cadences, and sometimes rose to the bugling of a gale. It
thrilled people and it melted them. Her eyes were wonderful.
Sometimes they rested on one person in the audience with a soft
and appealing look; then they gleamed and blazed with holy
passion. Her long arms with their fine tapering fingers—how they
helped to express her mind! But it was the face that was the great
exponent, and as emotions played on her own mobile features she
also touched the deep chords of every minister's heart. What struck
us most was the access she won to the hearts of penitents. The
mother-love in her was so deep and real that we all felt as if we,
too, could give her our sacred confidences. A favourite word of hers
was from St. Augustine, 'Love, and do as you like,' and every man in
our company felt she was a living illustration of it. Her beautiful and
choice language, simple, fresh, exquisitely fitting, and used with
superb ease and mastery, was a constant amazement. She never
attempted addresses or expositions, but her talks, for no other name
would she apply, were now and again gemmed with texts which
came as with a flash of diamonds, flaming."
Thus she revealed herself to men who know that the care of all
cares is the "cure" of souls—cura curarum cura animarum. She
warned them that the "apostolic life," the most Christ-like of all
vocations, is only for those who are willing to "fill up that which is
lacking of the sufferings of Christ." Prayer and fasting, love and
sacrifice, real asceticism combined with joyful enthusiasm are the
conditions of success in the never-ending warfare with evil. The
world will always be a broad field of battle. But the living Christ gives
so much of His real presence that His service is liberty and His
rewards are sure. No breath of human praise can compare with the
fervent and life-long gratitude which souls rescued from the powers
of darkness bear to their deliverer. Since the Maréchale laid aside the
French language—-perhaps only for a time—and resumed her
mother-tongue, she has received literally thousands of English letters
from both continents testifying to blessings received through her
ministry. I here give a few carefully selected extracts from these
letters with a few words of introduction.
One Sunday morning, as the Maréchale was about to address a
large congregation, the minister whispered to her: "You see those
two young ladies in black, if you can do anything with them, it would
be a miracle."
In one of the after-meetings of the mission, the Maréchale
approached the elder of these ladies and ventured to speak with her,
but intense reserve on her part made conversation impossible. A
cloud of utter despair seemed to have settled on her spirit. The look
in her eyes revealed sorrow too deep for words.
This is her story:
"At twelve o'clock one night I was returning home from business
with my mother and only sister. I found the body of my father
hanging in the corridor! I was so horrified that for a moment I could
not move, then, recovering my presence of mind, I put out the lights
and called my mother and sister to another door, just in time to
prevent them seeing the sight. But I can never forget it!
"Then my mother's health broke down, and for four years I
faithfully watched and cared for her.
 "During this second painful trial I received the startling news
that my dearest and only brother had met with a serious accident
while driving his own automobile. On arriving at the hospital in all
haste I was met with the words, 'Too late.' He was gone! The scene
which followed is too terrible for me to speak of. We adored him!
The effects of the shock hastened my mother's death. We said good-
bye to her, and oh, the recollection of it haunts me still.
"After my mother's death my sister and I left the house of
tragedy, broken down with sorrow and grief. These blows were
beyond my powers of endurance. In vain did I seek some ray of
comfort. Then I grew careless! Wine began to get a hold on me; and
I sank into depths of despair, of which you only know. I really
thought there was no God, and contemplated ending my own
existence.
"Through it all the Lord was looking down in tender compassion
and love. He sent you at this critical time in my sad career.
"When I look back on the past I can only praise God for what
He has done for me, through you.
"I am now conscious of the fact that He has washed and
redeemed me through the precious blood. Jesus is very dear to me.
He is the Lily of the Valley, the fairest of ten thousand to my soul.
The pleasures of the world have henceforth no attraction for me, in
Him I can overcome all temptations."

The following is from a gentleman in America, who, for thirty-five

years, had never entered a church. He happened to hear the
Maréchale once, and, rushing into the vestry, he broke down utterly
and told her the tragical story of his life.
 He was an illegitimate child, and his mother often used to beat
him until the blood ran. As a lad he struggled hard to be good, and,
although he was sometimes led astray through the drink, he always
shunned anything of an immoral character.
He married a sweet German girl, and by honest labour obtained
a very good position in New York, where he was esteemed by all
who knew him.
At this juncture he met the French woman who ruined his life.
He told the Maréchale between his sobs that his wife naturally
refused to have him back. Remorse and anguish of mind had driven
him twice to attempt suicide, but he was miraculously rescued. In
January, 1914, he writes:

"Dear Maréchale and honored Spiritual Mother:

"It was with joy that I received your dear letter. God has indeed
been wonderfully good to me, an undeserving sinner! He has rolled
away the stone from a heart heavy with sin and sorrow. I thank Him
daily for His mercy, and wonder how I could ever have lived so long
without Him. No wonder I failed and would have been lost, had I not
found Him at last through you, dear Maréchale. Life has a new
meaning for me. It is a pleasure to live now, and before it was a
curse. The musicians are playing ragtime while I am writing this, but
God is playing another melody in my heart. I thank Him for the
opportunity I have here to do good. God has indeed changed me.
He has given me great power over the minds of my men. The
change which, with His help, I have been able to effect in their
natures in two short weeks is wonderful to see. I am happy, very
happy.
"There is surely a devil, for he has sorely tempted me, but I
shake him off like a feather, smiling happily in my God-given
strength. I say to him, 'I fear you not, for I belong to Christ Jesus
forever.'
"I am indeed in an unholy place. It is given over to the devil in
every form, but what matters, I belong to Christ for all time.
"In prayer and humility I thank and greet thee, Beloved
Maréchale."

A beautiful girl in society writes the following:

"All night long I have whispered your name over and over again
to myself, saying, 'You have given me life, Maréchale, do you hear
me? LIFE!!!!' I was dying of remorse and fear. I shudder when I
think of what would have become of me if I had not come to you. I
used to say to myself, 'Oh, well, what's the use, I have sinned
beyond recall, so why not sin again, and again, and again? I am
destined for Hell anyway, I may just as well get there as fast as I
can'—that is what you have saved me from.
"I wonder how many times I have prayed 'Let me forget, only
let me forget,' and the more I would pray, the more I would
remember, and the more I would remember the more terrified I
would get, until life seemed to slip away from me, and I would fall
down, down, down, into a bottomless pit of horror.... And now I
LIVE!!!! Oh, Maréchale (how I love that name, it sounds like music
to me!) My mother gave life to my poor miserable body, but you
gave life to my soul. My mother would not mind me loving you so if
she knew that you gave me back to her."
Again she writes: "I have lived alone, absolutely alone. God only
knows how utterly alone I have been! But now I have Him, a Some
One who cares! Is it not wonderful, Maréchale, I am no longer alone
for I have my Christ? The warm thing that flames in my soul is the
knowledge that He loves me! Now I know why all these years I have
searched with empty hands for something. I have it now, I LIVE!!!!"

A young gentleman whose life was transformed through attending

the meetings held by the Maréchale's family in Keswick writes from
Beyrout, Syria:
"Oh! what a responsibility it is for us to be the ambassadors of
Christ, to represent Him to those who do not know Him, to be His
Images! If it were not for us Christians, who so often stand in His
way, Christ might have a chance. Some of the college men are
meeting daily in my room to pray and I wish you could hear them
praying in English, Arabic, Turkish, Armenian and even Abyssinian.
You would not understand the words, but you could never mistake
the spirit.
"To-day I invited a man whom I know is in the hold of a terrible
vice. He came, and during the half hour he looked as though he had
made a mistake in coming, but, before he left, he had led in prayer
for strength to overcome temptation.
"Don't you think America is a fine country? And yet, with all its
great resources, opportunities and phenomenal progress, it is a very
wicked country in many parts. Races, nations and individuals may
prosper and succeed in plans for betterment and still be without the
realisation of God,—they may be 'Good but Godless.'"

THE MARÉCHALE
(From a photograph taken at the
 Gainsborough Studio,
Oxford Street, London, W., in 1913)

With the tribute of one grateful heart this sketch may fitly close.
"I have been used in the past in the conversion of hundreds of
souls, but I made a compromise and it has spelt ruin to my soul. No
one knows how vile I have been, meriting desertion by God and
man.... I had resolved to end my existence, but somehow I was
brought to that meeting to hear about that Russian lady. Even then I
determined you should not influence me, but God somehow through
you gripped my life. I saw myself in the true light as (I say the
words not in their usual sense) a 'blasted hypocrite.' Don't forget to
echo and re-echo the words that reached me, 'Compromise with the
world spells ruin.' That burnt into my soul.... I remember while you
spoke a big lump rising in my throat, and just as you were closing
your address the thought came, 'I wonder if she would understand.'
Ay, more, I remember how you received me that day. God bless you.
I came out of hell. I have a clear sky. I want to let you know that the
consciousness of forgiveness of the past has come with almost an
overwhelming force, and an awful load has gone. No daughter ever
loved mother more than I love you, I know that. Why is it? Because
God made you the means of my salvation. My heart just bursts with
love and gratitude. So I am yours, and at that last great day you will
see it if I come through at last.... Dear one, have you ever thought
of this—some one by a gallant effort rescues lives from fire or
shipwreck; the world applauds and honours the deliverer. You (by
the grace of God) rescued me from shipwreck of soul. Christ will
own it before His Father and all the countless multitudes."

THE END

*** END OF THIS PROJECT GUTENBERG EBOOK THE MARÉCHALE

***
*** END OF THE PROJECT GUTENBERG EBOOK THE MARÉCHALE
(CATHERINE BOOTH-CLIBBORN) ***

Updated editions will replace the previous one—the old editions

will be renamed.

Creating the works from print editions not protected by U.S.

copyright law means that no one owns a United States
copyright in these works, so the Foundation (and you!) can copy
and distribute it in the United States without permission and
without paying copyright royalties. Special rules, set forth in the
General Terms of Use part of this license, apply to copying and
distributing Project Gutenberg™ electronic works to protect the
PROJECT GUTENBERG™ concept and trademark. Project
Gutenberg is a registered trademark, and may not be used if
you charge for an eBook, except by following the terms of the
trademark license, including paying royalties for use of the
Project Gutenberg trademark. If you do not charge anything for
copies of this eBook, complying with the trademark license is
very easy. You may use this eBook for nearly any purpose such
as creation of derivative works, reports, performances and
research. Project Gutenberg eBooks may be modified and
printed and given away—you may do practically ANYTHING in
the United States with eBooks not protected by U.S. copyright
law. Redistribution is subject to the trademark license, especially
commercial redistribution.

START: FULL LICENSE

THE FULL PROJECT GUTENBERG LICENSE
 PLEASE READ THIS BEFORE YOU DISTRIBUTE OR USE THIS WORK

To protect the Project Gutenberg™ mission of promoting the

free distribution of electronic works, by using or distributing this
work (or any other work associated in any way with the phrase
“Project Gutenberg”), you agree to comply with all the terms of
the Full Project Gutenberg™ License available with this file or
online at www.gutenberg.org/license.

Section 1. General Terms of Use and

Redistributing Project Gutenberg™
electronic works
1.A. By reading or using any part of this Project Gutenberg™
electronic work, you indicate that you have read, understand,
agree to and accept all the terms of this license and intellectual
property (trademark/copyright) agreement. If you do not agree
to abide by all the terms of this agreement, you must cease
using and return or destroy all copies of Project Gutenberg™
electronic works in your possession. If you paid a fee for
obtaining a copy of or access to a Project Gutenberg™
electronic work and you do not agree to be bound by the terms
of this agreement, you may obtain a refund from the person or
entity to whom you paid the fee as set forth in paragraph 1.E.8.

1.B. “Project Gutenberg” is a registered trademark. It may only

be used on or associated in any way with an electronic work by
people who agree to be bound by the terms of this agreement.
There are a few things that you can do with most Project
Gutenberg™ electronic works even without complying with the
full terms of this agreement. See paragraph 1.C below. There
are a lot of things you can do with Project Gutenberg™
electronic works if you follow the terms of this agreement and
help preserve free future access to Project Gutenberg™
electronic works. See paragraph 1.E below.
1.C. The Project Gutenberg Literary Archive Foundation (“the
Foundation” or PGLAF), owns a compilation copyright in the
collection of Project Gutenberg™ electronic works. Nearly all the
individual works in the collection are in the public domain in the
United States. If an individual work is unprotected by copyright
law in the United States and you are located in the United
States, we do not claim a right to prevent you from copying,
distributing, performing, displaying or creating derivative works
based on the work as long as all references to Project
Gutenberg are removed. Of course, we hope that you will
support the Project Gutenberg™ mission of promoting free
access to electronic works by freely sharing Project Gutenberg™
works in compliance with the terms of this agreement for
keeping the Project Gutenberg™ name associated with the
work. You can easily comply with the terms of this agreement
by keeping this work in the same format with its attached full
Project Gutenberg™ License when you share it without charge
with others.

1.D. The copyright laws of the place where you are located also
govern what you can do with this work. Copyright laws in most
countries are in a constant state of change. If you are outside
the United States, check the laws of your country in addition to
the terms of this agreement before downloading, copying,
displaying, performing, distributing or creating derivative works
based on this work or any other Project Gutenberg™ work. The
Foundation makes no representations concerning the copyright
status of any work in any country other than the United States.

1.E. Unless you have removed all references to Project

Gutenberg:

1.E.1. The following sentence, with active links to, or other

immediate access to, the full Project Gutenberg™ License must
appear prominently whenever any copy of a Project
Gutenberg™ work (any work on which the phrase “Project
Gutenberg” appears, or with which the phrase “Project
Gutenberg” is associated) is accessed, displayed, performed,
viewed, copied or distributed:

This eBook is for the use of anyone anywhere in

the United States and most other parts of the
world at no cost and with almost no restrictions
whatsoever. You may copy it, give it away or re-
use it under the terms of the Project Gutenberg
License included with this eBook or online at
www.gutenberg.org. If you are not located in the
United States, you will have to check the laws of
the country where you are located before using
this eBook.

1.E.2. If an individual Project Gutenberg™ electronic work is

derived from texts not protected by U.S. copyright law (does not
contain a notice indicating that it is posted with permission of
the copyright holder), the work can be copied and distributed to
anyone in the United States without paying any fees or charges.
If you are redistributing or providing access to a work with the
phrase “Project Gutenberg” associated with or appearing on the
work, you must comply either with the requirements of
paragraphs 1.E.1 through 1.E.7 or obtain permission for the use
of the work and the Project Gutenberg™ trademark as set forth
in paragraphs 1.E.8 or 1.E.9.

1.E.3. If an individual Project Gutenberg™ electronic work is

posted with the permission of the copyright holder, your use and
distribution must comply with both paragraphs 1.E.1 through
1.E.7 and any additional terms imposed by the copyright holder.
Additional terms will be linked to the Project Gutenberg™
License for all works posted with the permission of the copyright
holder found at the beginning of this work.
 1.E.4. Do not unlink or detach or remove the full Project
Gutenberg™ License terms from this work, or any files
containing a part of this work or any other work associated with
Project Gutenberg™.

1.E.5. Do not copy, display, perform, distribute or redistribute

this electronic work, or any part of this electronic work, without
prominently displaying the sentence set forth in paragraph 1.E.1
with active links or immediate access to the full terms of the
Project Gutenberg™ License.

1.E.6. You may convert to and distribute this work in any binary,
compressed, marked up, nonproprietary or proprietary form,
including any word processing or hypertext form. However, if
you provide access to or distribute copies of a Project
Gutenberg™ work in a format other than “Plain Vanilla ASCII” or
other format used in the official version posted on the official
Project Gutenberg™ website (www.gutenberg.org), you must,
at no additional cost, fee or expense to the user, provide a copy,
a means of exporting a copy, or a means of obtaining a copy
upon request, of the work in its original “Plain Vanilla ASCII” or
other form. Any alternate format must include the full Project
Gutenberg™ License as specified in paragraph 1.E.1.

1.E.7. Do not charge a fee for access to, viewing, displaying,

performing, copying or distributing any Project Gutenberg™
works unless you comply with paragraph 1.E.8 or 1.E.9.

1.E.8. You may charge a reasonable fee for copies of or

providing access to or distributing Project Gutenberg™
electronic works provided that:

• You pay a royalty fee of 20% of the gross profits you derive
from the use of Project Gutenberg™ works calculated using the
method you already use to calculate your applicable taxes. The
fee is owed to the owner of the Project Gutenberg™ trademark,
 but he has agreed to donate royalties under this paragraph to
the Project Gutenberg Literary Archive Foundation. Royalty
payments must be paid within 60 days following each date on
which you prepare (or are legally required to prepare) your
periodic tax returns. Royalty payments should be clearly marked
as such and sent to the Project Gutenberg Literary Archive
Foundation at the address specified in Section 4, “Information
about donations to the Project Gutenberg Literary Archive
Foundation.”

• You provide a full refund of any money paid by a user who

notifies you in writing (or by e-mail) within 30 days of receipt
that s/he does not agree to the terms of the full Project
Gutenberg™ License. You must require such a user to return or
destroy all copies of the works possessed in a physical medium
and discontinue all use of and all access to other copies of
Project Gutenberg™ works.

• You provide, in accordance with paragraph 1.F.3, a full refund of

any money paid for a work or a replacement copy, if a defect in
the electronic work is discovered and reported to you within 90
days of receipt of the work.

• You comply with all other terms of this agreement for free
distribution of Project Gutenberg™ works.

1.E.9. If you wish to charge a fee or distribute a Project

Gutenberg™ electronic work or group of works on different
terms than are set forth in this agreement, you must obtain
permission in writing from the Project Gutenberg Literary
Archive Foundation, the manager of the Project Gutenberg™
trademark. Contact the Foundation as set forth in Section 3
below.

1.F.
1.F.1. Project Gutenberg volunteers and employees expend
considerable effort to identify, do copyright research on,
transcribe and proofread works not protected by U.S. copyright
law in creating the Project Gutenberg™ collection. Despite these
efforts, Project Gutenberg™ electronic works, and the medium
on which they may be stored, may contain “Defects,” such as,
but not limited to, incomplete, inaccurate or corrupt data,
transcription errors, a copyright or other intellectual property
infringement, a defective or damaged disk or other medium, a
computer virus, or computer codes that damage or cannot be
read by your equipment.

1.F.2. LIMITED WARRANTY, DISCLAIMER OF DAMAGES - Except

for the “Right of Replacement or Refund” described in
paragraph 1.F.3, the Project Gutenberg Literary Archive
Foundation, the owner of the Project Gutenberg™ trademark,
and any other party distributing a Project Gutenberg™ electronic
work under this agreement, disclaim all liability to you for
damages, costs and expenses, including legal fees. YOU AGREE
THAT YOU HAVE NO REMEDIES FOR NEGLIGENCE, STRICT
LIABILITY, BREACH OF WARRANTY OR BREACH OF CONTRACT
EXCEPT THOSE PROVIDED IN PARAGRAPH 1.F.3. YOU AGREE
THAT THE FOUNDATION, THE TRADEMARK OWNER, AND ANY
DISTRIBUTOR UNDER THIS AGREEMENT WILL NOT BE LIABLE
TO YOU FOR ACTUAL, DIRECT, INDIRECT, CONSEQUENTIAL,
PUNITIVE OR INCIDENTAL DAMAGES EVEN IF YOU GIVE
NOTICE OF THE POSSIBILITY OF SUCH DAMAGE.

1.F.3. LIMITED RIGHT OF REPLACEMENT OR REFUND - If you

discover a defect in this electronic work within 90 days of
receiving it, you can receive a refund of the money (if any) you
paid for it by sending a written explanation to the person you
received the work from. If you received the work on a physical
medium, you must return the medium with your written
explanation. The person or entity that provided you with the
defective work may elect to provide a replacement copy in lieu
of a refund. If you received the work electronically, the person
or entity providing it to you may choose to give you a second
opportunity to receive the work electronically in lieu of a refund.
If the second copy is also defective, you may demand a refund
in writing without further opportunities to fix the problem.

1.F.4. Except for the limited right of replacement or refund set

forth in paragraph 1.F.3, this work is provided to you ‘AS-IS’,
WITH NO OTHER WARRANTIES OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO WARRANTIES OF
MERCHANTABILITY OR FITNESS FOR ANY PURPOSE.

1.F.5. Some states do not allow disclaimers of certain implied

warranties or the exclusion or limitation of certain types of
damages. If any disclaimer or limitation set forth in this
agreement violates the law of the state applicable to this
agreement, the agreement shall be interpreted to make the
maximum disclaimer or limitation permitted by the applicable
state law. The invalidity or unenforceability of any provision of
this agreement shall not void the remaining provisions.

1.F.6. INDEMNITY - You agree to indemnify and hold the

Foundation, the trademark owner, any agent or employee of the
Foundation, anyone providing copies of Project Gutenberg™
electronic works in accordance with this agreement, and any
volunteers associated with the production, promotion and
distribution of Project Gutenberg™ electronic works, harmless
from all liability, costs and expenses, including legal fees, that
arise directly or indirectly from any of the following which you
do or cause to occur: (a) distribution of this or any Project
Gutenberg™ work, (b) alteration, modification, or additions or
deletions to any Project Gutenberg™ work, and (c) any Defect
you cause.
 Section 2. Information about the Mission
of Project Gutenberg™
Project Gutenberg™ is synonymous with the free distribution of
electronic works in formats readable by the widest variety of
computers including obsolete, old, middle-aged and new
computers. It exists because of the efforts of hundreds of
volunteers and donations from people in all walks of life.

Volunteers and financial support to provide volunteers with the

assistance they need are critical to reaching Project
Gutenberg™’s goals and ensuring that the Project Gutenberg™
collection will remain freely available for generations to come. In
2001, the Project Gutenberg Literary Archive Foundation was
created to provide a secure and permanent future for Project
Gutenberg™ and future generations. To learn more about the
Project Gutenberg Literary Archive Foundation and how your
efforts and donations can help, see Sections 3 and 4 and the
Foundation information page at www.gutenberg.org.

Section 3. Information about the Project

Gutenberg Literary Archive Foundation
The Project Gutenberg Literary Archive Foundation is a non-
profit 501(c)(3) educational corporation organized under the
laws of the state of Mississippi and granted tax exempt status
by the Internal Revenue Service. The Foundation’s EIN or
federal tax identification number is 64-6221541. Contributions
to the Project Gutenberg Literary Archive Foundation are tax
deductible to the full extent permitted by U.S. federal laws and
your state’s laws.

The Foundation’s business office is located at 809 North 1500

West, Salt Lake City, UT 84116, (801) 596-1887. Email contact
links and up to date contact information can be found at the
Foundation’s website and official page at
www.gutenberg.org/contact

Section 4. Information about Donations to

the Project Gutenberg Literary Archive
Foundation
Project Gutenberg™ depends upon and cannot survive without
widespread public support and donations to carry out its mission
of increasing the number of public domain and licensed works
that can be freely distributed in machine-readable form
accessible by the widest array of equipment including outdated
equipment. Many small donations ($1 to $5,000) are particularly
important to maintaining tax exempt status with the IRS.

The Foundation is committed to complying with the laws

regulating charities and charitable donations in all 50 states of
the United States. Compliance requirements are not uniform
and it takes a considerable effort, much paperwork and many
fees to meet and keep up with these requirements. We do not
solicit donations in locations where we have not received written
confirmation of compliance. To SEND DONATIONS or determine
the status of compliance for any particular state visit
www.gutenberg.org/donate.

While we cannot and do not solicit contributions from states

where we have not met the solicitation requirements, we know
of no prohibition against accepting unsolicited donations from
donors in such states who approach us with offers to donate.

International donations are gratefully accepted, but we cannot

make any statements concerning tax treatment of donations
received from outside the United States. U.S. laws alone swamp
our small staff.
Please check the Project Gutenberg web pages for current
donation methods and addresses. Donations are accepted in a
number of other ways including checks, online payments and
credit card donations. To donate, please visit:
www.gutenberg.org/donate.

Section 5. General Information About

Project Gutenberg™ electronic works
Professor Michael S. Hart was the originator of the Project
Gutenberg™ concept of a library of electronic works that could
be freely shared with anyone. For forty years, he produced and
distributed Project Gutenberg™ eBooks with only a loose
network of volunteer support.

Project Gutenberg™ eBooks are often created from several

printed editions, all of which are confirmed as not protected by
copyright in the U.S. unless a copyright notice is included. Thus,
we do not necessarily keep eBooks in compliance with any
particular paper edition.

Most people start at our website which has the main PG search
facility: www.gutenberg.org.

This website includes information about Project Gutenberg™,

including how to make donations to the Project Gutenberg
Literary Archive Foundation, how to help produce our new
eBooks, and how to subscribe to our email newsletter to hear
about new eBooks.
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

ebookultra.com