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Calcium-Binding Protein Protocols
Volume II
M E T H O D S I N M O L E C U L A R B I O L O G YTM
John M. Walker, Series Editor
207.Human Antibodies for Cancer Therapy: Reviews and Proto- 178.Antibody Phage Display: Methods and Protocols, edited by
cols.edited by Martin Welschof and Jürgen Krauss, 2002 Philippa M. O’Brien and Robert Aitken, 2001
206.Endothelin Protocols, edited by Janet J. Maguire and Anthony 177.Two-Hybrid Systems: Methods and Protocols, edited by Paul
P. Davenport, 2002 N. MacDonald, 2001
205.E. coli Gene Expression Protocols, edited by Peter E. 176.Steroid Receptor Methods: Protocols and Assays, edited by
Vaillancourt, 2002 Benjamin A. Lieberman, 2001
204.Molecular Cytogenetics: Methods and Protocols, edited by 175.Genomics Protocols, edited by Michael P. Starkey and Ramnath
Yao-Shan Fan, 2002 Elaswarapu, 2001
203.In Situ Detection of DNA Damage: Methods and Protocols, 174.Epstein-Barr Virus Protocols, edited by Joanna B. Wilson and
edited by Vladimir V. Didenko, 2002 Gerhard H. W. May, 2001
202.Thyroid Hormone Receptors: Methods and Protocols, edited 173.Calcium-Binding Protein Protocols, Volume 2: Methods and
by Aria Baniahmad, 2002 Techniques,edited by Hans J. Vogel, 2001
201.Combinatorial Library Methods and Protocols, edited by 172.Calcium-Binding Protein Protocols, Volume 1: Reviews and
Lisa B. English, 2002 Case Histories, edited by Hans J. Vogel, 2001
200.DNA Methylation Protocols, edited by Ken I. Mills and 171.Proteoglycan Protocols, edited by Renato V. Iozzo, 2001
Bernie H, Ramsahoye, 2002 170.DNA Arrays: Methods and Protocols, edited by Jang B.
199.Liposome Methods and Protocols, edited by Subhash C. Basu Rampal, 2001
and Manju Basu, 2002 169.Neurotrophin Protocols, edited by Robert A. Rush, 2001
198.Neural Stem Cells: Methods and Protocols, edited by Tanja 168.Protein Structure, Stability, and Folding, edited by Kenneth
Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanberg, 2002 P. Murphy, 2001
197.Mitochondrial DNA: Methods and Protocols, edited by Will- 167.DNA Sequencing Protocols, Second Edition, edited by Colin
iam C. Copeland, 2002 A. Graham and Alison J. M. Hill, 2001
196.Oxidants and Antioxidants: Ultrastructural and Molecular 166.Immunotoxin Methods and Protocols, edited by Walter A.
Biology Protocols, edited by Donald Armstrong, 2002 Hall, 2001
195.Quantitative Trait Loci: Methods and Protocols,edited by 165.SV40 Protocols, edited by Leda Raptis, 2001
Nicola J. Camp and Angela Cox, 2002 164.Kinesin Protocols, edited by Isabelle Vernos, 2001
194.Post-translational Modification Reactions, edited by 163.Capillary Electrophoresis of Nucleic Acids, Volume 2:
Christoph Kannicht, 2002 Practical Applications of Capillary Electrophoresis, edited by
193.RT-PCR Protocols, edited by Joseph O’Connell, 2002 Keith R. Mitchelson and Jing Cheng, 2001
192.PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen 162.Capillary Electrophoresis of Nucleic Acids, Volume 1:
and Harry W. Janes, 2002 Introduction to the Capillary Electrophoresis of Nucleic Acids,
191.Telomeres and Telomerase:Methods and Protocols, edited edited by Keith R. Mitchelson and Jing Cheng, 2001
byJohn A. Double and Michael J. Thompson, 2002 161.Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001
190.High Throughput Screening: Methods and Protocols, edited 160.Nuclease Methods and Protocols, edited by Catherine H.
byWilliam P. Janzen, 2002 Schein, 2001
189.GTPase Protocols: The RAS Superfamily, edited by Edward 159.Amino Acid Analysis Protocols, edited by Catherine Cooper,
J. Manser and Thomas Leung, 2002 Nicole Packer, and Keith Williams, 2001
188.Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 158.Gene Knockoout Protocols, edited by Martin J. Tymms and
187.PCR Mutation Detection Protocols, edited by Bimal D. M. Ismail Kola, 2001
Theophilus and Ralph Rapley, 2002 157.Mycotoxin Protocols, edited by Mary W. Trucksess and Albert
186.Oxidative Stress and Antioxidant Protocols, edited by E. Pohland, 2001
Donald Armstrong, 2002 156.Antigen Processing and Presentation Protocols, edited by
185.Embryonic Stem Cells: Methods and Protocols, edited by Joyce C. Solheim, 2001
Kursad Turksen, 2002 155.Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000
184.Biostatistical Methods, edited by Stephen W. Looney, 2002 154.Connexin Methods and Protocols, edited by Roberto Bruzzone
183.Green Fluorescent Protein: Applications and Protocols, edited and Christian Giaume, 2001
byBarry W. Hicks, 2002 153.Neuropeptide Y Protocols, edited by Ambikaipakan
182.In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Balasubramaniam, 2000
Braman, 2002 152.DNA Repair Protocols: Prokaryotic Systems, edited by Patrick
181.Genomic Imprinting: Methods and Protocols, edited by Vaughan, 2000
Andrew Ward, 2002 151.Matrix Metalloproteinase Protocols, edited by Ian M. Clark,
180.Transgenesis Techniques, 2nd ed.: Principles and Protocols, 2001
edited by Alan R. Clarke, 2002 150.Complement Methods and Protocols, edited by B. Paul
179.Gene Probes: Principles and Protocols, edited by Marilena Morgan, 2000
Aquino de Muro and Ralph Rapley, 2002 149.The ELISA Guidebook,edited by John R. Crowther, 2000
M E T H O D S I N M O L E C U L A R B I O L O G YTM
Calcium-Binding
Protein
Protocols
Volume 2: Methods and Techniques
Edited by
Hans J. Vogel
Department of Biological Sciences, University of Calgary
Calgary, AB, Canada
Humana Press Totowa, New Jersey
© 2002 Humana Press Inc.
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Main entry under title: Methods in molecular biology™.
Calcium-binding protein protocols / edited by Hans J. Vogel
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Includes bibliographical references and index.
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ISBN 0-89603-689-8 (alk. paper)
1. Calcium-binding proteins--Research--Methodology I. Vogel, Hans J. II. Methods in molecular biology
(Clifton, N.J.) ; . v. 172, etc.
QP552.C24 C33 2001
572'.69—dc21 01-063354
Dedication
This book is dedicated to the memory of Dr. J. David Johnson (Columbus,
OH) whose untimely death on January 21, 2000 has deeply shocked all his
colleagues and friends. David has made numerous excellent contributions to
our understanding of calcium-binding proteins. His insight and enthusiasm
will be sadly missed.
Hans J. Vogel, PhD
v
Preface
Calcium plays an important role in a wide variety of biological processes.
This divalent metal ion can bind to a large number of proteins; by doing so it
modifies their biological activity or their stability. Because of its distinct chemi-
cal properties calcium is uniquely suited to act as an on–off switch or as a
light dimmer of biological activities. The two books entitled Calcium-Binding
Protein Protocols (Volumes I and II) focus on modern experimental analyses
and methodologies for the study of calcium-binding proteins. Both extracellu-
lar and intracellular calcium-binding proteins are discussed in detail. How-
ever, proteins involved in calcium handling (e.g., calcium pumps and calcium
channels), fall outside of the scope of these two volumes. Also, calcium-bind-
ing proteins involved in bone deposition will not be discussed, as this specific
topic has been addressed previously. The focus of these two books is on studies
of the calcium-binding proteins and their behavior in vitro and in vivo. The
primary emphasis is on protein chemistry and biophysical methods. Many of the
methods described will also be applicable to proteins that do not bind calcium.
Calcium-Binding Protein Protocols is divided into three main sections. The
section entitled Introduction and Reviews provides information on the role of
calcium in intracellular secondary messenger activation mechanisms. More-
over, unique aspects of calcium chemistry and the utilization of calcium in
dairy proteins, as well as calcium-binding proteins involved in blood clotting, are
addressed. The second section entitled Calcium-Binding Proteins: Case Studies
provides a wealth of information about protein purification and characterization
strategies, X-ray crystallography, and other studies that are focused on specific
calcium-binding proteins. Together, these two sections comprise Volume I of
this series. By introducing the various classes of intra- and extracellular cal-
cium-binding proteins and their modes of action, these two sections set the
stage and provide the necessary background for the third section. The final
section entitled Methods and Techniques to Study Calcium-Binding Proteins
makes up Volume II of Calcium-Binding Protein Protocols. Here the focus is
on the use of a range of modern experimental techniques that can be employed
to study the solution structure, stability, dynamics, calcium-binding proper-
ties, and biological activity of calcium-binding proteins in general. As well,
studies of their ligand-binding properties and their distribution in cells are
included. In addition to enzymatic assays and more routine spectroscopic and
protein chemistry techniques, particular attention has been paid in the second
volume to modern NMR approaches, thermodynamic analyses, kinetic mea-
vii
viii Preface
surements such as surface plasmon resonance, strategies for amino acid
sequence alignments, as well as fluorescence methods to study the distribu-
tion of calcium and calcium-binding proteins in cells. In preparing their chap-
ters, all the authors have attempted to share the little secrets that are
required to successfully apply these methods to related proteins. Together the
two volumes of Calcium-Binding Protein Protocols provide the reader with a
host of experimental methods that can be applied either to uncover new
aspects of earlier characterized calcium-binding proteins or to study newly
discovered proteins.
As more and more calcium-binding proteins are being uncovered through
genome sequencing efforts and protein interaction studies (e.g., affinity chro-
matography, crosslinking, or yeast two-hybrid systems) the time seemed right
to collect all the methods used to characterize these proteins in a book. The
methods detailed here should provide the reader with the essential tools for
their analysis in terms of structure, dynamics, and function. The hope is that
these two volumes will contribute to our understanding of the part of the pro-
teome, which relies on interactions with calcium to carry out its functions.
In closing, I would like to thank Margaret Tew for her invaluable assistance
with the editing and organization of these two books. Finally, I would like to
thank the authors of the individual chapters, who are all experts in this field,
for their cooperation in producing these two volumes in a timely fashion.
Hans J. Vogel, PhD
Contents
Dedication.........................................................................................................v
Preface ...........................................................................................................vii
Contents of Companion Volume...................................................................xiii
Contributors..................................................................................................xv
PART III. METHODS AND TECHNIQUES TO STUDY
CALCIUM-BINDING PROTEINS
1 Quantitative Analysis of Ca2+-Binding by Flow Dialysis
Michio Yazawa........................................................................................3
2 Calcium Binding to Proteins Studied via Competition
with Chromophoric Chelators
Sara Linse.............................................................................................15
3 Deconvolution of Calcium-Binding Curves: Facts and Fantasies
Jacques Haiech and Marie-Claude Kilhoffer....................................25
4 Absorption and Circular Dichroism Spectroscopy
Stephen R. Martin and Peter M. Bayley............................................43
5 Fourier Transform Infrared Spectroscopy
of Calcium-Binding Proteins
Heinz Fabian and Hans J. Vogel........................................................57
6 Steady-State Fluorescence Spectroscopy
Aalim M. Weljie and Hans J. Vogel....................................................75
7 Fluorescence Methods for Measuring Calcium Affinity and Calcium
Exchange with Proteins
J. David Johnson and Svetlana B. Tikunova...................................89
8 Surface Plasmon Resonance of Calcium-Binding Proteins
Karin Julenius....................................................................................103
9 Differential Scanning Calorimetry
Maria M. Lopez and George I. Makhatadze....................................113
10 Isothermal Titration Calorimetry
Maria M. Lopez and George I. Makhatadze....................................121
11 Multiangle Laser Light Scattering and Sedimentation Equilibrium
Leslie D. Hicks, Jean-René Alattia, Mitsuhiko Ikura,
and Cyril M. Kay.............................................................................127
ix
