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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,
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,
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
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,
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),
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
OWEN W. NADEAU • Department of Biochemistry and Molecular Biology,
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
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),
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
P. KEITH VERONESE • Department of Chemistry, The University of Alabama
xviii Contributors
OLEG K. VOROV • Department of Physics and Optical Science, University of North

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

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).
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
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
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I remember well that, throughout his preliminary ordeal, young
Smethick bore himself like one who had given up all hope of refuting
the terrible charges brought against him, and, I must say, the
formidable number of witnesses which the police brought up against
him more than explained that attitude.
Of course, Haggett was not called, but, as it happened, there were
plenty of people to swear that Mr. Laurence Smethick was seen
loitering round the gates of Clevere Hall after the guests had
departed on Christmas Eve. The head gardener, who lives at the
lodge, actually spoke to him, and Captain Glynne, leaning out of his
brougham window, was heard to exclaim:
“Hello, Smethick, what are you doing here at this time of night?”
And there were others, too.
To Captain Glynne’s credit, be it here recorded, he tried his best to
deny having recognised his unfortunate friend in the dark. Pressed
by the magistrate, he said obstinately:
“I thought at the time that it was Mr. Smethick standing by the
lodge gates, but on thinking the matter over I feel sure that I was
mistaken.”
On the other hand, what stood dead against young Smethick was,
firstly, the question of the ring, and then the fact that he was seen in
the immediate neighbourhood of Clevere, both at midnight and
again at about two, when some men, who had been on the watch
for the cattle-maimers, saw him walking away rapidly in the direction
of Pakethorpe.
What was, of course, unexplainable and very terrible to witness
was Mr. Smethick’s obstinate silence with regard to his own
movements during those fatal hours on that night. He did not
contradict those who said that they had seen him at about midnight
near the gates of Clevere, nor his own valet’s statements as to the
hour when he returned home. All he said was that he could not
account for what he did between the time when the guests left the
Hall and he himself went back to Pakethorpe. He realised the danger
in which he stood, and what caused him to be silent about a matter
which might mean life or death to him could not easily be
conjectured.
The ownership of the ring he could not and did not dispute. He
had lost it in the grounds of Clevere, he said. But the jeweller in
Coney Street swore that he had sold the ring to Mr. Smethick on the
18th of December, whilst it was a well-known and an admitted fact
that the young man had not openly been inside the gates of Clevere
for over a fortnight before that.
On this evidence Laurence Smethick was committed for trial.
Though the actual weapon with which the unfortunate Major had
been stabbed had not been found, nor its ownership traced, there
was such a vast array of circumstantial evidence against the young
man that bail was refused.
He had, on the advice of his solicitor, Mr. Grayson—one of the
ablest lawyers in York—reserved his defence, and on that miserable
afternoon at the close of the year, we all filed out of the crowded
court, feeling terribly depressed and anxious.
4
My dear lady and I walked back to our hotel in silence. Our hearts
seemed to weigh heavily within us. We felt mortally sorry for that
good-looking young Yorkshireman, who, we were convinced, was
innocent, yet at the same time seemed involved in a tangled web of
deadly circumstances from which he seemed quite unable to
extricate himself.
We did not feel like discussing the matter in the open streets,
neither did we make any comment when presently, in a block in the
traffic in Coney Street, we saw Margaret Ceely driving her smart
dog-cart, whilst sitting beside her, and talking with great earnestness
close to her ear, sat Captain Glynne.
She was in deep mourning, and had obviously been doing some
shopping, for she was surrounded with parcels; so perhaps it was
hypercritical to blame her. Yet somehow it struck me that just at the
moment when there hung in the balance the life and honour of a
man with whose name her own had oft been linked by popular
rumour, it showed more than callous contempt for his welfare to be
seen driving about with another man who, since his sudden access
to fortune, had undoubtedly become a rival in her favours.
When we arrived at the “Black Swan,” we were surprised to hear
that Mr. Grayson had called to see my dear lady, and was upstairs
waiting.
Lady Molly ran up to our sitting-room and greeted him with
marked cordiality. Mr. Grayson is an elderly, dry-looking man, but he
looked visibly affected, and it was some time before he seemed able
to plunge into the subject which had brought him hither. He fidgeted
in his chair, and started talking about the weather.
“I am not here in a strictly professional capacity, you know,” said
Lady Molly presently, with a kindly smile and with a view to helping
him out of his embarrassment. “Our police, I fear me, have an
exaggerated view of my capacities, and the men here asked me
unofficially to remain in the neighbourhood and to give them my
advice if they should require it. Our chief is very lenient to me, and
has allowed me to stay. Therefore, if there is anything I can do——”
“Indeed, indeed there is!” ejaculated Mr. Grayson with sudden
energy. “From all I hear, there is not another soul in the kingdom but
you who can save this innocent man from the gallows.”
My dear lady heaved a little sigh of satisfaction. She had all along
wanted to have a more important finger in that Yorkshire pie.
“Mr. Smethick?” she said.
“Yes; my unfortunate young client,” replied the lawyer. “I may as
well tell you,” he resumed after a slight pause, during which he
seemed to pull himself together, “as briefly as possible what
occurred on December 24th last and on the following Christmas
morning. You will then understand the terrible plight in which my
client finds himself, and how impossible it is for him to explain his
actions on that eventful night. You will understand, also, why I have
come to ask your help and your advice. Mr. Smethick considered
himself engaged to Miss Ceely. The engagement had not been made
public because of Major Ceely’s anticipated opposition, but the
young people had been very intimate, and many letters had passed
between them. On the morning of the 24th Mr. Smethick called at
the Hall, his intention then being merely to present his fiancée with
the ring you know of. You remember the unfortunate contretemps
that occurred: I mean the unprovoked quarrel sought by Major Ceely
with my poor client, ending with the irascible old man forbidding Mr.
Smethick the house.
“My client walked out of Clevere feeling, as you may well imagine,
very wrathful; on the doorstep, just as he was leaving, he met Miss
Margaret, and told her very briefly what had occurred. She took the
matter very lightly at first, but finally became more serious, and
ended the brief interview with the request that, since he could not
come to the dance after what had occurred, he should come and see
her afterwards, meeting her in the gardens soon after midnight. She
would not take the ring from him then, but talked a good deal of
sentiment about Christmas morning, asking him to bring the ring to
her at night, and also the letters which she had written him. Well—
you can guess the rest.”
Lady Molly nodded thoughtfully.
“Miss Ceely was playing a double game,” continued Mr. Grayson,
earnestly. “She was determined to break off all relationship with Mr.
Smethick, for she had transferred her volatile affections to Captain
Glynne, who had lately become heir to an earldom and £40,000 a
year. Under the guise of sentimental twaddle she got my unfortunate
client to meet her at night in the grounds of Clevere and to give up
to her the letters which might have compromised her in the eyes of
her new lover. At two o’clock a.m. Major Ceely was murdered by one
of his numerous enemies; as to which I do not know, nor does Mr.
Smethick. He had just parted from Miss Ceely at the very moment
when the first cry of ‘Murder’ roused Clevere from its slumbers. This
she could confirm if she only would, for the two were still in sight of
each other, she inside the gates, he just a little way down the road.
Mr. Smethick saw Margaret Ceely run rapidly back towards the
house. He waited about a little while, half hesitating what to do;
then he reflected that his presence might be embarrassing, or even
compromising to her whom, in spite of all, he still loved dearly; and
knowing that there were plenty of men in and about the house to
render what assistance was necessary, he finally turned his steps
and went home a broken-hearted man, since she had given him the
go-by, taken her letters away, and flung contemptuously into the
mud the ring he had bought for her.”
The lawyer paused, mopping his forehead and gazing with whole-
souled earnestness at my lady’s beautiful, thoughtful face.
“Has Mr. Smethick spoken to Miss Ceely since?” asked Lady Molly,
after a while.
“No; but I did,” replied the lawyer.
“What was her attitude?”
“One of bitter and callous contempt. She denies my unfortunate
client’s story from beginning to end; declares that she never saw him
after she bade him ‘good-morning’ on the doorstep of Clevere Hall,
when she heard of his unfortunate quarrel with her father. Nay,
more; she scornfully calls the whole tale a cowardly attempt to
shield a dastardly crime behind a still more dastardly libel on a
defenceless girl.”
We were all silent now, buried in thought which none of us would
have cared to translate into words. That the impasse seemed indeed
hopeless no one could deny.
The tower of damning evidence against the unfortunate young
man had indeed been built by remorseless circumstances with no
faltering hand.
Margaret Ceely alone could have saved him, but with brutal
indifference she preferred the sacrifice of an innocent man’s life and
honour to that of her own chances of a brilliant marriage. There are
such women in the world; thank God I have never met any but that
one!
Yet am I wrong when I say that she alone could save the
unfortunate young man, who throughout was behaving with such
consummate gallantry, refusing to give his own explanation of the
events that occurred on that Christmas morning, unless she chose
first to tell the tale. There was one present now in the dingy little
room at the “Black Swan” who could disentangle that weird skein of
coincidences, if any human being not gifted with miraculous powers
could indeed do it at this eleventh hour.
She now said, gently:
“What would you like me to do in this matter, Mr. Grayson? And
why have you come to me rather than to the police?”
“How can I go with this tale to the police?” he ejaculated in
obvious despair. “Would they not also look upon it as a dastardly
libel on a woman’s reputation? We have no proofs, remember, and
Miss Ceely denies the whole story from first to last. No, no!” he
exclaimed with wonderful fervour. “I came to you because I have
heard of your marvellous gifts, your extraordinary intuition. Someone
murdered Major Ceely! It was not my old friend Colonel Smethick’s
son. Find out who it was, then! I beg of you, find out who it was!”
He fell back in his chair, broken down with grief. With inexpressible
gentleness Lady Molly went up to him and placed her beautiful white
hand on his shoulder.
“I will do my best, Mr. Grayson,” she said simply.
5
We remained alone and singularly quiet the whole of that evening.

That my dear lady’s active brain was hard at work I could guess by
the brilliance of her eyes, and that sort of absolute stillness in her
person through which one could almost feel the delicate nerves
vibrating.
The story told her by the lawyer had moved her singularly. Mind
you, she had always been morally convinced of young Smethick’s
innocence, but in her the professional woman always fought hard
battles against the sentimentalist, and in this instance the
overwhelming circumstantial evidence and the conviction of her
superiors had forced her to accept the young man’s guilt as
something out of her ken.
By his silence, too, the young man had tacitly confessed; and if a
man is perceived on the very scene of a crime, both before it has
been committed and directly afterwards; if something admittedly
belonging to him is found within three yards of where the murderer
must have stood; if, added to this, he has had a bitter quarrel with
the victim, and can give no account of his actions or whereabouts
during the fatal time, it were vain to cling to optimistic beliefs in that
same man’s innocence.
But now matters had assumed an altogether different aspect. The
story told by Mr. Smethick’s lawyer had all the appearance of truth.
Margaret Ceely’s character, her callousness on the very day when
her late fiancé stood in the dock, her quick transference of her
affections to the richer man, all made the account of the events on
Christmas night as told by Mr. Grayson extremely plausible.
No wonder my dear lady was buried in thought.
“I shall have to take the threads up from the beginning, Mary,” she
said to me the following morning, when after breakfast she
appeared in her neat coat and skirt, with hat and gloves, ready to go
out, “so, on the whole, I think I will begin with a visit to the
Haggetts.”
“I may come with you, I suppose?” I suggested meekly.
“Oh, yes!” she rejoined carelessly.
Somehow I had an inkling that the carelessness of her mood was
only on the surface. It was not likely that she—my sweet, womanly,
ultra-feminine, beautiful lady—should feel callously on this absorbing
subject.
We motored down to Bishopthorpe. It was bitterly cold, raw,
damp, and foggy. The chauffeur had some difficulty in finding the
cottage, the “home” of the imbecile gardener and his wife.
There was certainly not much look of home about the place.
When, after much knocking at the door, Mrs. Haggett finally opened
it, we saw before us one of the most miserable, slatternly places I
think I ever saw.
In reply to Lady Molly’s somewhat curt inquiry, the woman said
that Haggett was in bed, suffering from one of his “fits.”
“That is a great pity,” said my dear lady, rather unsympathetically,
I thought, “for I must speak with him at once.”
“What is it about?” asked the woman, sullenly. “I can take a
message.”
“I am afraid not,” rejoined my lady. “I was asked to see Haggett
personally.”
“By whom, I’d like to know,” she retorted, now almost insolently.
“I dare say you would. But you are wasting precious time. Hadn’t
you better help your husband on with his clothes? This lady and I
will wait in the parlour.”
After some hesitation the woman finally complied, looking very
sulky the while.
We went into the miserable little room wherein not only grinding
poverty but also untidiness and dirt were visible all round. We sat
down on two of the cleanest-looking chairs, and waited whilst a
colloquy in subdued voices went on in the room over our heads.
The colloquy, I may say, seemed to consist of agitated whispers
on one part, and wailing complaints on the other. This was followed
presently by some thuds and much shuffling, and presently Haggett,
looking uncared-for, dirty, and unkempt, entered the parlour,
followed by his wife.
He came forward, dragging his ill-shod feet and pulling nervously
at his forelock.
“Ah!” said my lady, kindly; “I am glad to see you down, Haggett,
though I am afraid I haven’t very good news for you.”
“Yes, miss!” murmured the man, obviously not quite
comprehending what was said to him.
“I represent the workhouse authorities,” continued Lady Molly,
“and I thought we could arrange for you and your wife to come into
the Union to-night, perhaps.”
“The Union?” here interposed the woman, roughly. “What do you
mean? We ain’t going to the Union?”
“Well! but since you are not staying here,” rejoined my lady,
blandly, “you will find it impossible to get another situation for your
husband in his present mental condition.”
“Miss Ceely won’t give us the go-by,” she retorted defiantly.
“She might wish to carry out her late father’s intentions,” said
Lady Molly with seeming carelessness.
“The Major was a cruel, cantankerous brute,” shouted the woman
with unpremeditated violence. “Haggett had served him faithfully for
twelve years, and——”
She checked herself abruptly, and cast one of her quick, furtive
glances at Lady Molly.
Her silence now had become as significant as her outburst of rage,
and it was Lady Molly who concluded the phrase for her.
“And yet he dismissed him without warning,” she said calmly.
“Who told you that?” retorted the woman.
“The same people, no doubt, who declare that you and Haggett
had a grudge against the Major for this dismissal.”
“That’s a lie,” asserted Mrs. Haggett, doggedly; “we gave
information about Mr. Smethick having killed the Major because——”
“Ah,” interrupted Lady Molly, quickly, “but then Mr. Smethick did
not murder Major Ceely, and your information therefore was
useless!”
“Then who killed the Major, I should like to know?”
Her manner was arrogant, coarse, and extremely unpleasant. I
marvelled why my dear lady put up with it, and what was going on
in that busy brain of hers. She looked quite urbane and smiling,
whilst I wondered what in the world she meant by this story of the
workhouse and the dismissal of Haggett.
“Ah, that’s what none of us know!” she now said lightly; “some
folks say it was your husband.”
“They lie!” she retorted quickly, whilst the imbecile, evidently not
understanding the drift of the conversation, was mechanically
stroking his red mop of hair and looking helplessly all round him.
“He was home before the cries of ‘Murder’ were heard in the
house,” continued Mrs. Haggett.
“How do you know?” asked Lady Molly, quickly.
“How do I know?”
“Yes; you couldn’t have heard the cries all the way to this cottage
—why, it’s over half a mile from the Hall!”
“He was home, I say,” she repeated with dogged obstinacy.
“You sent him?”
“He didn’t do it——”
“No one will believe you, especially when the knife is found.”
“What knife?”
“His clasp knife, with which he killed Major Ceely,” said Lady Molly,
quietly; “see, he has it in his hand now.”
And with a sudden, wholly unexpected gesture she pointed to the
imbecile, who in an aimless way had prowled round the room whilst
this rapid colloquy was going on.
The purport of it all must in some sort of way have found an echo
in his enfeebled brain. He wandered up to the dresser whereon lay
the remnants of that morning’s breakfast, together with some
crockery and utensils.
In that same half-witted and irresponsible way he had picked up
one of the knives and now was holding it out towards his wife, whilst
a look of fear spread over his countenance.
“I can’t do it, Annie, I can’t—you’d better do it,” he said.
There was dead silence in the little room. The woman Haggett
stood as if turned to stone. Ignorant and superstitious as she was, I
suppose that the situation had laid hold of her nerves, and that she
felt that the finger of a relentless Fate was even now being pointed
at her.
The imbecile was shuffling forward, closer and closer to his wife,
still holding out the knife towards her and murmuring brokenly:
“I can’t do it. You’d better, Annie—you’d better——”
He was close to her now, and all at once her rigidity and nerve-
strain gave way; she gave a hoarse cry, and snatching the knife from
the poor wretch, she rushed at him ready to strike.
Lady Molly and I were both young, active and strong; and there
was nothing of the squeamish grande dame about my dear lady
when quick action was needed. But even then we had some
difficulty in dragging Annie Haggett away from her miserable
husband. Blinded with fury, she was ready to kill the man who had
betrayed her. Finally, we succeeded in wresting the knife from her.
You may be sure that it required some pluck after that to sit down
again quietly and to remain in the same room with this woman, who
already had one crime upon her conscience, and with this weird,
half-witted creature who kept on murmuring pitiably:
“You’d better do it, Annie——”
Well, you’ve read the account of the case, so you know what
followed. Lady Molly did not move from that room until she had
obtained the woman’s full confession. All she did for her own
protection was to order me to open the window and to blow the
police whistle which she handed to me. The police-station
fortunately was not very far, and sound carried in the frosty air.
She admitted to me afterwards that it had been foolish, perhaps,
not to have brought Etty or Danvers with her, but she was supremely
anxious not to put the woman on the alert from the very start,
hence her circumlocutory speeches anent the workhouse, and
Haggett’s probable dismissal.
That the woman had had some connection with the crime, Lady
Molly, with her keen intuition, had always felt; but as there was no
witness to the murder itself, and all circumstantial evidence was
dead against young Smethick, there was only one chance of
successful discovery, and that was the murderer’s own confession.
If you think over the interview between my dear lady and the
Haggetts on that memorable morning, you will realise how admirably
Lady Molly had led up to the weird finish. She would not speak to
the woman unless Haggett was present, and she felt sure that as
soon as the subject of the murder cropped up, the imbecile would
either do or say something that would reveal the truth.
Mechanically, when Major Ceely’s name was mentioned, he had
taken up the knife. The whole scene recurred to his tottering mind.
That the Major had summarily dismissed him recently was one of
those bold guesses which Lady Molly was wont to make.
That Haggett had been merely egged on by his wife, and had
been too terrified at the last to do the deed himself was no surprise
to her, and hardly one to me, whilst the fact that the woman
ultimately wreaked her own passionate revenge upon the
unfortunate Major was hardly to be wondered at, in the face of her
own coarse and elemental personality.
Cowed by the quickness of events, and by the appearance of
Danvers and Etty on the scene, she finally made full confession.
She was maddened by the Major’s brutality, when with rough,
cruel words he suddenly turned her husband adrift, refusing to give
him further employment. She herself had great ascendency over the
imbecile, and had drilled him into a part of hate and of revenge. At
first he had seemed ready and willing to obey. It was arranged that
he was to watch on the terrace every night until such time as an
alarm of the recurrence of the cattle-maiming outrages should lure
the Major out alone.
This effectually occurred on Christmas morning, but not before
Haggett, frightened and pusillanimous, was ready to flee rather than
to accomplish the villainous deed. But Annie Haggett, guessing
perhaps that he would shrink from the crime at the last, had also
kept watch every night. Picture the prospective murderer watching
and being watched!
When Haggett came across his wife he deputed her to do the
deed herself.
I suppose that either terror of discovery or merely desire for the
promised reward had caused the woman to fasten the crime on
another.
The finding of the ring by Haggett was the beginning of that cruel
thought which, but for my dear lady’s marvellous powers, would
indeed have sent a brave young man to the gallows.
Ah, you wish to know if Margaret Ceely is married? No! Captain
Glynne cried off. What suspicions crossed his mind I cannot say; but
he never proposed to Margaret, and now she is in Australia—staying
with an aunt, I think—and she has sold Clevere Hall.
VIII.
THE BAG OF SAND
Of course, I knew at once by the expression of her face that

morning that my dear lady had some important business on hand.
She had a bundle in her arms, consisting of a shabby-looking coat
and skirt, and a very dowdy hat trimmed with bunches of cheap,
calico roses.
“Put on these things at once, Mary,” she said curtly, “for you are
going to apply for the situation of ‘good plain cook,’ so mind you look
the part.”
“But where in the world——?” I gasped in astonishment.
“In the house of Mr. Nicholas Jones, in Eaton Terrace,” she
interrupted dryly, “the one occupied until recently by his sister, the
late Mrs. Dunstan. Mrs. Jones is advertising for a cook, and you
must get that place.”
As you know, I have carried obedience to the level of a fine art.
Nor was I altogether astonished that my dear lady had at last been
asked to put one of her dainty fingers in that Dunstan pie, which
was puzzling our fellows more completely than any other case I have
ever known.
I don’t know if you remember the many circumstances, the
various contradictions which were cropping up at every turn, and
which baffled our ablest detectives at the very moment when they
thought themselves most near the solution of that strange mystery.
Mrs. Dunstan herself was a very uninteresting individual: self-
righteous, self-conscious and fat, a perfect type of the moneyed
middle-class woman whose balance at the local bank is invariably
heavier than that of her neighbours. Her niece, Violet Frostwicke,
lived with her: a smart, pretty girl, inordinately fond of dainty clothes
and other luxuries which money can give. Being totally impecunious
herself, she bore with the older woman’s constantly varying caprices
with almost angelic patience, a fact probably attributable to Mrs.
Dunstan’s testamentary intentions, which, as she often averred,
were in favour of her niece.
In addition to these two ladies, the household consisted of three
servants and Miss Cruikshank. The latter was a quiet, unassuming
girl who was by way of being secretary and lady-help to Mrs.
Dunstan, but who, in reality, was nothing but a willing drudge. Up
betimes in the morning, she combined the work of a housekeeper
with that of an upper servant. She interviewed the tradespeople,
kept the servants in order, and ironed and smartened up Miss Violet’s
blouses. A Cinderella, in fact.
Mrs. Dunstan kept a cook and two maids, all of whom had been
with her for years. In addition to these, a charwoman came very
early in the morning to light fires, clean boots, and do the front
steps.
On November 22nd, 1907—for the early history of this curious
drama dates back to that year—the charwoman who had been
employed at Mrs. Dunstan’s house in Eaton Terrace for some
considerable time, sent word in the morning that in future she would
be unable to come. Her husband had been obliged to move to
lodgings nearer to his work, and she herself could not undertake to
come the greater distance at the early hour at which Mrs. Dunstan
required her.
The woman had written a very nice letter explaining these facts,
and sent it by hand, stating at the same time that the bearer of the
note was a very respectable woman, a friend of her own, who would
be very pleased to “oblige” Mrs. Dunstan by taking on the morning’s
work.
I must tell you that the message and its bearer arrived at Eaton
Terrace somewhere about 6.0 a.m., when no one was down except
the Cinderella of the house, Miss Cruikshank.
She saw the woman, liked her appearance, and there and then
engaged her to do the work, subject to Mrs. Dunstan’s approval.
The woman, who had given her name as Mrs. Thomas, seemed
very quiet and respectable. She said that she lived close by, in St.
Peter’s Mews, and therefore could come as early as Mrs. Dunstan
wished. In fact, from that day, she came every morning at 5.30 a.m.,
and by seven o’clock had finished her work, and was able to go
home.
If, in addition to these details, I tell you that, at that time, pretty
Miss Violet Frostwicke was engaged to a young Scotsman, Mr. David
Athol, of whom her aunt totally disapproved, I shall have put before
you all the personages who, directly or indirectly, were connected
with that drama, the final act of which has not yet been witnessed
either by the police or by the public.
2
On the following New Year’s Eve, Mrs. Dunstan, as was her invariable
custom on that day, went to her married brother’s house to dine and
to see the New Year in.
During her absence the usual thing occurred at Eaton Terrace.
Miss Violet Frostwicke took the opportunity of inviting Mr. David
Athol to spend the evening with her.
Mrs. Dunstan’s servants, mind you, all knew of the engagement
between the young people, and with the characteristic sentimentality
of their class, connived at these secret meetings and helped to
hoodwink the irascible old aunt.
Mr. Athol was a good-looking young man, whose chief demerit lay
in his total lack of money or prospects. Also he was by way of being
an actor, another deadly sin in the eyes of the puritanically-minded
old lady.
Already, on more than one occasion, there had been vigorous
wordy warfare ’twixt Mr. Athol and Mrs. Dunstan, and the latter had
declared that if Violet chose to take up with this mountebank, she
should never see a penny of her aunt’s money now or in the future.
The young man did not come very often to Eaton Terrace, but on
this festive New Year’s Eve, when Mrs. Dunstan was not expected to
be home until long after midnight, it seemed too splendid an
opportunity for an ardent lover to miss.
As ill-luck would have it, Mrs. Dunstan had not felt very well after
her copious dinner, and her brother, Mr. Nicholas Jones, escorted her
home soon after ten o’clock.
Jane, the parlour-maid who opened the front door, was, in her
own graphic language, “knocked all of a heap” when she saw her
mistress, knowing full well that Mr. Athol was still in the dining-room
with Miss Violet, and that Miss Cruikshank was at that very moment
busy getting him a whisky and soda.
Meanwhile the coat and hat in the hall had revealed the young
man’s presence in the house.
For a moment Mrs. Dunstan paused, whilst Jane stood by
trembling with fright. Then the old lady turned to Mr. Nicholas Jones,
who was still standing on the doorstep, and said quietly:
“Will you telephone over to Mr. Blenkinsop, Nick, the first thing in
the morning, and tell him I’ll be at his office by ten o’clock?”
Mr. Blenkinsop was Mrs. Dunstan’s solicitor, and as Jane explained
to the cook later on, what could such an appointment mean but a
determination to cut Miss Violet out of the missis’s will with the
proverbial shilling?
After this Mrs. Dunstan took leave of her brother and went
straight into the dining-room.
According to the subsequent testimony of all three servants, the
mistress “went on dreadful.” Words were not easily distinguishable
from behind the closed door, but it seems that, immediately she
entered, Mrs. Dunstan’s voice was raised as if in terrible anger, and a
few moments later Miss Violet fled crying from the dining-room, and
ran quickly upstairs.
Whilst the door was thus momentarily opened and shut, the voice
of the old lady was heard saying, in majestic wrath:
“That’s what you have done. Get out of this house. As for her,
she’ll never see a penny of my money, and she may starve for aught
I care!”
The quarrel seems to have continued for a short while after that,
the servants being too deeply awed by those last vindictive words
which they had heard to take much note of what went on
subsequently.
Mrs. Dunstan and Mr. Athol were closeted together for some time;
but apparently the old lady’s wrath did not subside, for when she
marched up to bed an hour later she was heard to say:
“Out of this house she shall go, and the first thing in the morning,
too. I’ll have no goings-on with a mountebank like you.”
Miss Cruikshank was terribly upset.
“It is a frightful blow for Miss Violet,” she said to cook, “but
perhaps Mrs. Dunstan will feel more forgiving in the morning. I’ll
take her up a glass of champagne now. She is very fond of that, and
it will help her to get to sleep.”
Miss Cruikshank went up with the champagne, and told cook to
see Mr. Athol out of the house; but the young man, who seemed
very anxious and agitated, would not go away immediately. He
stayed in the dining-room, smoking, for a while, and when the two
younger servants went up to bed, he asked cook to let him remain
until he had seen Miss Violet once more, for he was sure she would
come down again—he had asked Miss Cruikshank to beg her to do
so.
Mrs. Kennett, the cook, was a kind-hearted old woman. She had
taken the young people under her special protection, and felt very
vexed that the course of true love should not be allowed to run quite
smoothly. So she told Mr. Athol to make himself happy and
comfortable in the dining-room, and she would sit up by the fire in
the library until he was ready to go.
The good soul thereupon made up the fire in the library, drew a
chair in front of it, and—went fast to sleep.
Suddenly something awoke her. She sat up and looked round in
that dazed manner peculiar to people just aroused from deep sleep.
She looked at the clock; it was past three. Surely, she thought, it
must have been Mr. Athol calling to her which had caused her to
wake. She went into the hall, where the gas had not yet been turned
off, and there she saw Miss Violet, fully dressed and wearing a hat
and coat, in the very act of going out at the front door.
In the cook’s own words, before she could ask a question or even
utter a sound, the young girl had opened the front door, which was
still on the latch, and then banged it to again, she herself having
disappeared into the darkness of the street beyond.
Mrs. Kennett ran to the door and out into the street as fast as her
old legs would let her; but the night was an exceptionally foggy one.
Violet, no doubt, had walked rapidly away, and there came no
answer to Mrs. Kennett’s repeated calls.
Thoroughly upset, and not knowing what to do, the good woman
went back into the house. Mr. Athol had evidently left, for there was
no sign of him in the dining-room or elsewhere. She then went
upstairs and knocked at Mrs. Dunstan’s door. To her astonishment
the gas was still burning in her mistress’s room, as she could see a
thin ray of light filtering through the keyhole. At her first knock there
came a quick, impatient answer:
“What is it?”
“Miss Violet, ’m,” said the cook, who was too agitated to speak
very coherently, “she is gone——”
“The best thing she could do,” came promptly from the other side
of the door. “You go to bed, Mrs. Kennett, and don’t worry.”
Whereupon the gas was suddenly turned off inside the room, and,
in spite of Mrs. Kennett’s further feeble protests, no other word
issued from the room save another impatient:
“Go to bed.”
The cook then did as she was bid; but before going to bed she
made the round of the house, turned off all the gas, and finally
bolted the front door.
3
Some three hours later the servants were called, as usual, by Miss
Cruikshank, who then went down to open the area door to Mrs.
Thomas, the charwoman.
At half-past six, when Mary the housemaid came down, candle in
hand, she saw the charwoman a flight or two lower down, also
apparently in the act of going downstairs. This astonished Mary not
a little, as the woman’s work lay entirely in the basement, and she
was supposed never to come to the upper floors.
The woman, though walking rapidly down the stairs, seemed,
moreover, to be carrying something heavy.
“Anything wrong, Mrs. Thomas?” asked Mary, in a whisper.
The woman looked up, pausing a moment immediately under the
gas bracket, the by-pass of which shed a feeble light upon her and
upon her burden. The latter Mary recognised as the bag containing
the sand which, on frosty mornings, had to be strewn on the front
steps of the house.
On the whole, though she certainly was puzzled, Mary did not
think very much about the incident then. As was her custom, she
went into the housemaid’s closet, got the hot water for Miss
Cruikshank’s bath, and carried it to the latter’s room, where she also
pulled up the blinds and got things ready generally. For Miss
Cruikshank usually ran down in her dressing-gown, and came up to
tidy herself later on.
As a rule, by the time the three servants got downstairs, it was
nearly seven, and Mrs. Thomas had generally gone by that time; but
on this occasion Mary was earlier. Miss Cruikshank was busy in the
kitchen getting Mrs. Dunstan’s tea ready. Mary spoke about seeing
Mrs. Thomas on the stairs with the bag of sand, and Miss
Cruikshank, too, was very astonished at the occurrence.
Mrs. Kennett was not yet down, and the charwoman apparently
had gone; her work had been done as usual, and the sand was
strewn over the stone steps in front, as the frosty fog had rendered
them very slippery.
At a quarter past seven Miss Cruikshank went up with Mrs.
Dunstan’s tea, and less than two minutes later a fearful scream rang
through the entire house, followed by the noise of breaking crockery.
In an instant the two maids ran upstairs, straight to Mrs.
Dunstan’s room, the door of which stood wide open.
The first thing Mary and Jane were conscious of was a terrific
smell of gas, then of Miss Cruikshank, with eyes dilated with horror,
staring at the bed in front of her, whereon lay Mrs. Dunstan, with
one end of a piece of indiarubber piping still resting in her mouth,
her jaw having dropped in death. The other end of that piece of
piping was attached to the burner of a gas-bracket on the wall close
by.
Every window in the room was fastened and the curtains drawn.
The whole room reeked of gas.
Mrs. Dunstan had been asphyxiated by its fumes.
4
A year went by after the discovery of the mysterious tragedy, and I

can assure you that our fellows at the Yard had one of the toughest
jobs in connection with the case that ever fell to their lot. Just think
of all the contradictions which met them at every turn.
Firstly, the disappearance of Miss Violet.
No sooner had the women in the Dunstan household roused
themselves sufficiently from their horror at the terrible discovery
which they had just made, than they were confronted with another
almost equally awful fact—awful, of course, because of its
connection with the primary tragedy.
Miss Violet Frostwicke had gone. Her room was empty, her bed
had not been slept in. She herself had been seen by the cook, Mrs.
Kennett, stealing out of the house at dead of night.
To connect the pretty, dainty young girl even remotely with a
crime so hideous, so callous, as the deliberate murder of an old
woman, who had been as a mother to her, seemed absolutely out of
the question, and by tacit consent the four women, who now
remained in the desolate and gloom-laden house at Eaton Terrace,
forbore to mention Miss Violet Frostwicke’s name either to police or
doctor.
Both these, of course, had been summoned immediately; Miss
Cruikshank sending Mary to the police-station and thence to Dr.
Folwell, in Eaton Square, whilst Jane went off in a cab to fetch Mr.
Nicholas Jones, who, fortunately, had not yet left for his place of
business.
The doctor’s and the police-inspector’s first thought, on examining
the mise en scène of the terrible tragedy, was that Mrs. Dunstan had
committed suicide. It was practically impossible to imagine that a
woman in full possession of health and strength would allow a piece
of indiarubber piping to be fixed between her teeth, and would,
without a struggle, continue to inhale the poisonous fumes which
would mean certain death. Yet there were no marks of injury upon
the body, nothing to show how sufficient unconsciousness had been
produced in the victim to permit of the miscreant completing his
awesome deed.
But the theory of suicide set up by Dr. Folwell was promptly
refuted by the most cursory examination of the room.
Though the drawers were found closed, they had obviously been
turned over, as if the murderer had been in search either of money
or papers, or the key of the safe.
The latter, on investigation, was found to be open, whilst the key
lay on the floor close by. A brief examination of the safe revealed the
fact that the tin boxes must have been ransacked, for they contained
neither money nor important papers now, whilst the gold and
platinum settings of necklaces, bracelets, and a tiara showed that
the stones—which, as Mr. Nicholas Jones subsequently averred, were
of considerable value—had been carefully, if somewhat clumsily,
taken out by obviously inexperienced hands.
On the whole, therefore, appearances suggested deliberate,
systematic, and very leisurely robbery, which wholly contradicted the
theory of suicide.
Then suddenly the name of Miss Frostwicke was mentioned. Who
first brought it on the tapis no one subsequently could say; but in a
moment the whole story of the young girl’s engagement to Mr. Athol,
in defiance of her aunt’s wishes, the quarrel of the night before, and
the final disappearance of both young people from the house during
the small hours of the morning, was dragged from the four unwilling
witnesses by the able police-inspector.
Nay, more. One very unpleasant little circumstance was detailed
by one of the maids and corroborated by Miss Cruikshank.
It seems that when the latter took up the champagne to Mrs.
Dunstan, the old lady desired Miss Violet to come to her room. Mary,
the housemaid, was on the stairs when she saw the young girl, still
dressed in her evening gown of white chiffon, her eyes still swollen
with tears, knocking at her aunt’s door.
The police-inspector was busy taking notes, already building up in
his mind a simple, if very sensational, case against Violet Frostwicke,
when Mrs. Kennett promptly upset all his calculations.
Miss Violet could have had nothing to do with the murder of her
aunt, seeing that Mrs. Dunstan was alive and actually spoke to the
cook when the latter knocked at her bedroom door after she had
seen the young girl walk out of the house.
Then came the question of Mr. Athol. But, if you remember, it was
quite impossible even to begin to build up a case against the young
man. His own statement that he left the house at about midnight,
having totally forgotten to rouse the cook when he did so, was
amply corroborated from every side.
The cabman who took him up to the corner of Eaton Terrace at
11.50 p.m. was one witness in his favour; his landlady at his rooms
in Jermyn Street, who let him in, since he had mislaid his latchkey,
and who took him up some tea at seven o’clock the next morning,
was another; whilst, when Mary saw Miss Violet going into her aunt’s
room, the clock at St. Peter’s, Eaton Square, was just striking twelve.
I dare say you think I ought by now to have mentioned the
charwoman, Mrs. Thomas, who represented the final, most
complete, most hopeless contradiction in this remarkable case.
Mrs. Thomas was seen by Mary, the housemaid, at half-past six
o’clock in the morning, coming down from the upper floors, where
she had no business to be, and carrying the bag of sand used for
strewing over the slippery front-door steps.
The bag of sand, of course, was always kept in the area.
The moment that bag of sand was mentioned Dr. Folwell gave a
curious gasp. Here, at least, was the solution to one mystery. The
victim had been stunned whilst still in bed by a blow on the head
dealt with that bag of sand; and whilst she was unconscious the
callous miscreant had robbed her and finally asphyxiated her with
the gas fumes.
Where was the woman who, at half-past six in the morning, was
seen in possession of the silent instrument of death?
Mrs. Thomas had disappeared. The last that was then or ever has
been seen of her was when she passed underneath the dim light of
a by-pass on the landing, as if tired out with the weight which she
was carrying.
Since then, as you know, the police have been unswerving in their
efforts to find Mrs. Thomas. The address which she had given in St.
Peter’s Mews was found to be false. No one of that name or
appearance had ever been seen there.
The woman who was supposed to have sent her with a letter of
recommendation to Mrs. Dunstan knew nothing of her. She swore
that she had never sent anyone with a letter to Mrs. Dunstan. She
gave up her work there one day because she found it too hard at
such an early hour in the morning; but she never heard anything
more from her late employer after that.
Strange, wasn’t it, that two people should have disappeared out of
that house on that same memorable night?
Of course, you will remember the tremendous sensation that was
caused some twenty-four hours later, when it transpired that the
young person who had thrown herself into the river from Waterloo
Bridge on that same eventful morning, and whose body was
subsequently recovered and conveyed to the Thames Police station,
was identified as Miss Violet Frostwicke, the niece of the lady who
had been murdered in her own house in Eaton Terrace.
Neither money nor diamonds were found on poor Miss Violet. She
had herself given the most complete proof that she, at least, had no
hand in robbing or killing Mrs. Dunstan.
The public wondered why she took her aunt’s wrath and her
probable disinheritance so fearfully to heart, and sympathised with
Mr. David Athol for the terribly sad loss which he had sustained.
But Mrs. Thomas, the charwoman, had not yet been found.
5
I think I looked an extremely respectable, good plain cook when I

presented myself at the house in Eaton Terrace in response to the
advertisement in the “Daily Telegraph.”
As, in addition to my prepossessing appearance, I also asked very
low wages and declared myself ready to do anything except scour
the front steps and the stone area, I was immediately engaged by
Mrs. Jones, and was duly installed in the house the following day
under the name of Mrs. Curwen.
But few events had occurred here since the discovery of the dual
tragedy, now more than a year ago, and none that had thrown any
light upon the mystery which surrounded it.
The verdict at the inquest had been one of wilful murder against a
person known as Mrs. Thomas, the weight of evidence, coupled with
her disappearance, having been very heavy against her; and there
was a warrant out for her arrest.
Mrs. Dunstan had died intestate. To the astonishment of all those
in the know, she had never signed the will which Messrs. Blenkinsop
and Blenkinsop had drafted for her, and wherein she bequeathed
£20,000 and the lease of her house in Eaton Terrace to her beloved
niece, Violet Frostwicke, £1,000 to Miss Cruikshank, and other,
smaller, legacies to friends or servants.
In default of a will, Mr. Nicholas Jones, only brother of the
deceased, became possessed of all her wealth.
He was a very rich man himself, and many people thought that he
ought to give Miss Cruikshank the £1,000 which the poor girl had
thus lost through no fault of her own.
What his ultimate intentions were with regard to this no one could
know. For the present he contented himself with moving to Eaton
Terrace with his family; and, as his wife was a great invalid, he
asked Miss Cruikshank to continue to make her home in the house
and to help in its management.
Neither the diamonds nor the money stolen from Mrs. Dunstan’s
safe were ever traced. It seems that Mrs. Dunstan, a day or two
before her death, had sold a freehold cottage which she owned near
Teddington. The money, as is customary, had been handed over to
her in gold, in Mr. Blenkinsop’s office, and she had been foolish
enough not to bank it immediately. This money and the diamonds
had been the chief spoils of her assailant. And all the while no trace
of Mrs. Thomas, in spite of the most strenuous efforts on the part of
the police to find her.
Strangely enough, when I had been in Eaton Terrace about three
days, and was already getting very tired of early rising and hard
work, the charwoman there fell ill one day and did not come to her
work as usual.
I, of course, grumbled like six, for I had to be on my hands and
knees the next morning scrubbing stone steps, and my thoughts of
Lady Molly, for the moment, were not quite as loyal as they usually
were.
Suddenly I heard a shuffling footstep close behind me. I turned
and saw a rough-looking, ill-dressed woman standing at the bottom
of the steps.
“What do you want?” I asked sourly, for I was in a very bad
humour.
“I saw you scrubbing them steps, miss,” she replied in a raucous
voice; “my ’usband is out of work, and the children hain’t ’ad no
breakfast this morning. I’d do them steps, miss, if you’d give me a
trifle.”
The woman certainly did not look very prepossessing, with her
shabby, broad-brimmed hat hiding the upper part of her face, and
her skirt, torn and muddy, pinned up untidily round her stooping
figure.
However, I did not think that I could be doing anything very wrong
by letting her do this one bit of rough work, which I hated, so I
agreed to give her sixpence, and left her there with kneeling mat
and scrubbing-brush, and went in, leaving, however, the front door
open.
In the hall I met Miss Cruikshank, who, as usual, was down before
everybody else.
“What is it, Curwen?” she asked, for through the open door she
had caught sight of the woman kneeling on the step.
“A woman, miss,” I replied, somewhat curtly. “She offered to do
the steps. I thought Mrs. Jones wouldn’t mind, as Mrs. Callaghan
hasn’t turned up.”
Miss Cruikshank hesitated an instant, and then walked up to the
front door.
At the same moment the woman looked up, rose from her knees,
and boldly went up to accost Miss Cruikshank.
“You’ll remember me, miss,” she said, in her raucous voice. “I
used to work for Mrs. Dunstan once. My name is Mrs. Thomas.”
No wonder Miss Cruikshank uttered a quickly smothered cry of
horror. Thinking that she would faint, I ran to her assistance; but she
waved me aside and then said quite quietly:
“This poor woman’s mind is deranged. She is no more Mrs.
Thomas than I am. Perhaps we had better send for the police.”
“Yes, miss; p’r’aps you’d better,” said the woman with a sigh. “My
secret has been weighin’ heavy on me of late.”
“But, my good woman,” said Miss Cruikshank, very kindly, for I
suppose that she thought, as I did, that this was one of those
singular cases of madness which sometimes cause innocent people
to accuse themselves of undiscovered crimes. “You are not Mrs.
Thomas at all. I knew Mrs. Thomas well, of course—and——”
“Of course you knew me, miss,” replied the woman. “The last
conversation you and I had together was in the kitchen that
morning, when Mrs. Dunstan was killed. I remember your saying to
me——”
“Fetch the police, Curwen,” said Miss Cruikshank, peremptorily.
Whereupon the woman broke into a harsh and loud laugh of
defiance.
To tell you the truth, I was not a little puzzled. That this scene had
been foreseen by my dear lady, and that she had sent me to this
house on purpose that I should witness it, I was absolutely
convinced. But—here was my dilemma: ought I to warn the police at
once or not?
On the whole, I decided that my best plan would undoubtedly be
to communicate with Lady Molly first of all, and to await her
instructions. So I ran upstairs, scribbled a hasty note to my dear
lady, and, in response to Miss Cruikshank’s orders, flew out of the
house through the area gate, noticing, as I did so, that Miss
Cruikshank was still parleying with the woman on the doorstep.
I sent the note off to Maida Vale by taxicab; then I went back to
Eaton Terrace. Miss Cruikshank met me at the front door, and told
me that she had tried to detain the woman, pending my return; but
that she felt very sorry for the unfortunate creature, who obviously
was labouring under a delusion, and she had allowed her to go
away.
About an hour later I received a curt note from Lady Molly
ordering me to do nothing whatever without her special
authorisation.
In the course of the day, Miss Cruikshank told me that she had
been to the police-station, and had consulted with the inspector,
who said there would be no harm in engaging the pseudo Mrs.
Thomas to work at Eaton Terrace, especially as thus she would
remain under observation.
Then followed a curious era in Mr. Nicholas Jones’s otherwise well-
ordered household. We three servants, instead of being called at six
as heretofore, were allowed to sleep on until seven. When we came
down we were not scolded. On the contrary, we found our work
already done.
The charwoman—whoever she was—must have been a very hard-
working woman. It was marvellous what she accomplished single-
handed before seven a.m., by which time she had invariably gone.
The two maids, of course, were content to let this pleasant state
of things go on, but I was devoured with curiosity.
One morning I crept quietly downstairs and went into the kitchen
soon after six. I found the pseudo Mrs. Thomas sitting at a very
copious breakfast. I noticed that she had on altogether different—
though equally shabby and dirty—clothes from those she had worn

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# Springer ScienceþBusiness Media, LLC 2012

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

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

Kansas City, KS, USA Aron W. Fenton

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

This work was supported in part by NIH grant DK78076.

PART I MONITORING ALLOSTERIC FUNCTION

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

PART II MONITORING ALLOSTERIC CONFORMATIONAL CHANGES

7 A Review of Methods Used for Identifying Structural Changes

PART III MONITORING ALLOSTERIC CHANGES IN PROTEIN

PART IV MACROMOLECULAR AND LIGAND ENGINEERING

16 An In Vivo Approach to Isolating Allosteric Pathways

PART V COMPUTATIONAL METHODS/AIDS IN THE STUDY OF ALLOSTERY

21 A Critical Evaluation of Correlated Mutation Algorithms

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

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

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

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

Monitoring Allosteric Function

Binding Techniques to Study the Allosteric Energy Cycle

It is challenging to succinctly summarize the term “allostery”

We now have a wealth of experimental data indicating proteins

unliganded state of the protein and 1 denotes the same transition

The ensemble of conformations available to the protein in

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

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

DGapp ðXYÞ ¼ RT ln Kapp

Practically speaking, cooperativity is a thermodynamic

when both ligands are present via thermodynamic cycles, and to

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

The essentials of CD spectroscopy have been exhaustively elabo-

We remained alone and singularly quiet the whole of that evening.

Of course, I knew at once by the expression of her face that

A year went by after the discovery of the mysterious tragedy, and I

I think I looked an extremely respectable, good plain cook when I

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