calcium-binding protein protocols volume 2

Methods in Molecular Biology TM VOLUME 173 Calcium-Binding Protein Protocols Volume II Methods and Techniques Edited by Hans J Vogel HUMANA PRESS Calcium-Binding Protein Protocols Volume II METHODS IN MOLECULAR BIOLOGY TM John M Walker, Series Editor 207 Human Antibodies for Cancer Therapy: Reviews and Protocols edited by Martin Welschof and Jürgen Krauss, 2002 206 Endothelin Protocols, edited by Janet J Maguire and Anthony P Davenport, 2002 205 E coli Gene Expression Protocols, edited by Peter E Vaillancourt, 2002 204 Molecular Cytogenetics: Methods and Protocols, edited by Yao-Shan Fan, 2002 203 In Situ Detection of DNA Damage: Methods and Protocols, edited by Vladimir V Didenko, 2002 202 Thyroid Hormone Receptors: Methods and Protocols, edited by Aria Baniahmad, 2002 201 Combinatorial Library Methods and Protocols, edited by Lisa B English, 2002 200 DNA Methylation Protocols, edited by Ken I Mills and Bernie H, Ramsahoye, 2002 199 Liposome Methods and Protocols, edited by Subhash C Basu and Manju Basu, 2002 198 Neural Stem Cells: Methods and Protocols, edited by Tanja Zigova, Juan R Sanchez-Ramos, and Paul R Sanberg, 2002 197 Mitochondrial DNA: Methods and Protocols, edited by William C Copeland, 2002 196 Oxidants and Antioxidants: Ultrastructural and Molecular Biology Protocols, edited by Donald Armstrong, 2002 195 Quantitative Trait Loci: Methods and Protocols, edited by Nicola J Camp and Angela Cox, 2002 194 Post-translational Modification Reactions, edited by Christoph Kannicht, 2002 193 RT-PCR Protocols, edited by Joseph O’Connell, 2002 192 PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen and Harry W Janes, 2002 191 Telomeres and Telomerase: Methods and Protocols, edited by John A Double and Michael J Thompson, 2002 190 High Throughput Screening: Methods and Protocols, edited by William P Janzen, 2002 189 GTPase Protocols: The RAS Superfamily, edited by Edward J Manser and Thomas Leung, 2002 188 Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 187 PCR Mutation Detection Protocols, edited by Bimal D M Theophilus and Ralph Rapley, 2002 186 Oxidative Stress and Antioxidant Protocols, edited by Donald Armstrong, 2002 185 Embryonic Stem Cells: Methods and Protocols, edited by Kursad Turksen, 2002 184 Biostatistical Methods, edited by Stephen W Looney, 2002 183 Green Fluorescent Protein: Applications and Protocols, edited by Barry W Hicks, 2002 182 In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Braman, 2002 181 Genomic Imprinting: Methods and Protocols, edited by Andrew Ward, 2002 180 Transgenesis Techniques, 2nd ed.: Principles and Protocols, edited by Alan R Clarke, 2002 179 Gene Probes: Principles and Protocols, edited by Marilena Aquino de Muro and Ralph Rapley, 2002 178 Antibody Phage Display: Methods and Protocols, edited by Philippa M O’Brien and Robert Aitken, 2001 177 Two-Hybrid Systems: Methods and Protocols, edited by Paul N MacDonald, 2001 176 Steroid Receptor Methods: Protocols and Assays, edited by Benjamin A Lieberman, 2001 175 Genomics Protocols, edited by Michael P Starkey and Ramnath Elaswarapu, 2001 174 Epstein-Barr Virus Protocols, edited by Joanna B Wilson and Gerhard H W May, 2001 173 Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J Vogel, 2001 172 Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J Vogel, 2001 171 Proteoglycan Protocols, edited by Renato V Iozzo, 2001 170 DNA Arrays: Methods and Protocols, edited by Jang B Rampal, 2001 169 Neurotrophin Protocols, edited by Robert A Rush, 2001 168 Protein Structure, Stability, and Folding, edited by Kenneth P Murphy, 2001 167 DNA Sequencing Protocols, Second Edition, edited by Colin A Graham and Alison J M Hill, 2001 166 Immunotoxin Methods and Protocols, edited by Walter A Hall, 2001 165 SV40 Protocols, edited by Leda Raptis, 2001 164 Kinesin Protocols, edited by Isabelle Vernos, 2001 163 Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R Mitchelson and Jing Cheng, 2001 162 Capillary Electrophoresis of Nucleic Acids, Volume 1: Introduction to the Capillary Electrophoresis of Nucleic Acids, edited by Keith R Mitchelson and Jing Cheng, 2001 161 Cytoskeleton Methods and Protocols, edited by Ray H Gavin, 2001 160 Nuclease Methods and Protocols, edited by Catherine H Schein, 2001 159 Amino Acid Analysis Protocols, edited by Catherine Cooper, Nicole Packer, and Keith Williams, 2001 158 Gene Knockoout Protocols, edited by Martin J Tymms and Ismail Kola, 2001 157 Mycotoxin Protocols, edited by Mary W Trucksess and Albert E Pohland, 2001 156 Antigen Processing and Presentation Protocols, edited by Joyce C Solheim, 2001 155 Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000 154 Connexin Methods and Protocols, edited by Roberto Bruzzone and Christian Giaume, 2001 153 Neuropeptide Y Protocols , edited by Ambikaipakan Balasubramaniam, 2000 152 DNA Repair Protocols: Prokaryotic Systems, edited by Patrick Vaughan, 2000 151 Matrix Metalloproteinase Protocols, edited by Ian M Clark, 2001 150 Complement Methods and Protocols, edited by B Paul Morgan, 2000 149 The ELISA Guidebook, edited by John R Crowther, 2000 METHODS IN MOLECULAR BIOLOGY 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 TM © 2002 Humana Press Inc 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher Methods in Molecular Biology™ is a trademark of The Humana Press Inc The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents This publication is printed on acid-free paper ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials Cover design by Patricia F Cleary Cover illustration: From Fig 1A in Chapter 3, Vol “Crystal Structure of Calpain and Insights into Ca2+-Dependent Activation” by Zongchao Jia, Christopher M Hosfield, Peter L Davies, and John S Elce Production Editor: Kim Hoather-Potter For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: humana@humanapr.com, or visit our Website at www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923 For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc The fee code for users of the Transactional Reporting Service is: [0-89603-689-8/02 $10.00 + $00.25] Printed in the United States of America 10 Library of Congress Cataloging in Publication Data Main entry under title: Methods in molecular biology™ Calcium-binding protein protocols / edited by Hans J Vogel p cm (Methods in molecular biology; v v 172-) Includes bibliographical references and index Contents: v Reviews and case studies ISBN 0-89603-689-8 (alk paper) 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 chemical 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 extracellular and intracellular calcium-binding proteins are discussed in detail However, proteins involved in calcium handling (e.g., calcium pumps and calcium channels), fall outside of the scope of these two volumes Also, calcium-binding 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 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 Moreover, 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 calcium-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 properties, 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 distribution of calcium and calcium-binding proteins in cells In preparing their chapters, 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 chromatography, 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 proteome, 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 Quantitative Analysis of Ca2+-Binding by Flow Dialysis Michio Yazawa Calcium Binding to Proteins Studied via Competition with Chromophoric Chelators Sara Linse 15 Deconvolution of Calcium-Binding Curves: Facts and Fantasies Jacques Haiech and Marie-Claude Kilhoffer 25 Absorption and Circular Dichroism Spectroscopy Stephen R Martin and Peter M Bayley 43 Fourier Transform Infrared Spectroscopy of Calcium-Binding Proteins Heinz Fabian and Hans J Vogel 57 Steady-State Fluorescence Spectroscopy Aalim M Weljie and Hans J Vogel 75 Fluorescence Methods for Measuring Calcium Affinity and Calcium Exchange with Proteins J David Johnson and Svetlana B Tikunova 89 Surface Plasmon Resonance of Calcium-Binding Proteins Karin Julenius 103 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 402 Török et al Fig 14 Localization of FL-dextran calmodulin during mitosis µM of FL-dextran was microinjected and the cells were inspected under the same conditions as for FL-calmodulin FL-dextran reports distributions in the cytoplasmic water space and perhaps nonspecific binding FL-dextran localizes cytoplasmically in comparison to FL-calmodulin, which binds specifically to the astral tubule array Also, the localization of FL-dextran is far less well-defined, which suggests that binding of cellular structures is less specific the two events, TA- and FL-calmodulin are both applied to the same cell and their fluorescence emissions are scanned simultaneously The fluorescence of FL-calmodulin microinjected into sea urchin eggs (final concentration µM) is relatively insensitive to Ca2+ and target protein binding, but provides information on localization Figure 15 shows a sequence of mitotic transitions in a sea urchin egg microinjected with FL-calmodulin TA-calmodulin (final concentration of 10 µM) microinjected into the sea urchin eggs shows a 10-fold rise in fluorescence intensity on Ca2+ and target protein binding Figure 16 shows a sequence of mitotic transitions in a sea urchin egg microinjected with TA-calmodulin Note the fluorescence intensity differences between FL-calmodulin and TA-calmodulin Spatial Distribution of Ca2+-Binding Proteins 403 Fig 15 Indicator of calmodulin localization using FL-calmodulin The sequence shows mitotic events from 79 –121 where upon cell cleavage occurs The fluorescence of FL-calmodulin (final concentration µM) microinjected into sea urchin eggs is relatively insensitive to Ca2+ and target protein binding, but provides information on localization Simultaneous use of the Ca 2+ -sensitive (TA-calmodulin) and insensitive (FL-calmodulin) derivative allow us to distinguish between Ca 2+ activation of calmodulin and local concentration changes of calmodulin (see Fig 17) To test that calmodulin activation is required for mitotic transitions Trp peptide (potent calmodulin inhibitor) (12,13) was injected prior to fertilization Trp peptide blocks NEB and if injected postNEB blocks the metaphase-anaphase transition (17) This further suggests that Ca2+-calmodulin-dependent processes are required for mitotic transitions Notes The three important factors that must be observed while reacting 5-DTAF with calmodulin are pH, Ca 2+ (divalent cation) concentration, and no increase in reaction time If these factors are not observed carefully, the ratio of singly labeled 404 Török et al Fig 16 Indicator of calmodulin activation and localization using TA-calmodulin TA-calmodulin (final concentration of 10 µM) microinjected into sea urchin eggs shows a 10-fold rise in fluorescence intensity on Ca2+ and target protein binding The sequence shows the mitotic events from 79–121 FL-calmodulin (5-DTAF labeled on Lys75 of calmodulin) to doubly labeled FL-calmodulin (5-DTAF labeled on Lys75 and Lys 148 of calmodulin) will be reduced We select singly-labeled FL-calmodulin so that precise measurements of fluorescence can be made during imaging Both Lys75 and Lys77 are located in a relatively exposed region of calmodulin The fact that Lys77 is not very reactive compared to Lys75, originates from its relatively high pKa value (3) In both singly and doubly labeled FL-calmodulin, peak 11 was fluorescent and analysis by electrospray and nanospray mass spectrometry identified the peptide as fragment T2–T3 (residues 14 – 30) with some labeling on Lys21 Other minor fluorescent peaks that were not identified may represent a small amount of labeling on the other lysines or other peptide fragments containing the labeled Lys75 For imaging of spatial distribution of proteins, it is advantageous if the fluorophore covalently attached to calmodulin is not environmentally sensitive Spatial Distribution of Ca2+-Binding Proteins 405 Fig 17 Indicator of calmodulin activation using TA-calmodulin and FLcalmodulin Simultaneous use of the activation-sensitive and activation-insensitive derivative allows us to distinguish between activation and local concentration changes of calmodulin The sequence shows mitotic events from 79 –121 FL-calmodulin is an inert fluorophore If the [Ca2+] is changed from 10 nM – 0.1 mM at physiological ionic strength and pH, the fluorescence intensity of FL-calmodulin changes by less than 5% No further change occurs on peptide target binding Similar observations were made with Cy5-calmodulin and Texas Red-calmodulin Thus FL-, Cy5-, and Texas Red-calmodulins report calmodulin localization in the cell In contrast, if calmodulin is labeled at Lys75 with the environmentally sensitive TA-Cl probe (11), Ca2+-binding results in a 5.5-fold increase of fluorescence intensity and target binding may cause a further twofold increase (12) TA-calmodulin fluorescence thus reports the interactions of calmodulin, as well as its concentration changes in the cell In order to distinguish between the two events, TA- and FL-calmodulin can both be applied to the same cell and their fluorescence emissions are scanned simultaneously It is instructive to compare the target binding and enzyme activation properties of fluorescently labeled calmodulin with unlabeled calmodulin TA-calmodulin and 406 Török et al the significantly less-bright Lys75-labeled DANSYL-calmodulin bind to targets with an approx threefold increased dissociation constant and act as an activator of cyclic-AMP phosphodiesterase similar to unmodified calmodulin Lys75-modified calmodulins appear to act as a competitive inhibitor of smooth muscle myosin light-chain kinase (13) They do, however, activate calmodulin-dependent protein kinase II auto- and substrate phosphorylation (Török, K and Fraser, C., unpublished data) It is thus expected that Lys75-labeled calmodulins are accurate reporters of calmodulin movements and activities in the cell The inhibitory property can either be taken advantage of or countered by trace-level application of the fluorescent calmodulin in the cell References Cohen, P and Klee, C B., eds (1988) Calmodulin Elsevier, New York Mann, D and Vanaman, T C (1987) Specific chemical modification as a probe of calmodulin function Methods Enzymol 139, 417–433 Zhang, M and Vogel, H J (1993) NMR studies of the pKa’s of the lysine sidechains in calmodulin J Biol Chem 268, 22,420 – 22,428 Török, K., Lane, A N., Martin, S R., Janot, J.-M., and Bayley, P M (1992) Effects of calcium binding on the internal dynamic properties of bovine brain calmodulin, studied by NMR and optical spectroscopy Biochemistry 31, 3452 –3462 Giedroc, D P., Puett, D., Sinha, S K., and Brew, K (1987) Calcium effects on calmodulin lysine reactivities Arch Biochem Biophys 252, 136–144 Selsted, M E (1997) HPLC methods for purification of antimicrobial peptides Methods Mol Biol 78, 17–33 Smith, R D., Loo, J A., Edmonds, C G., Barinaga, C J., and Udseth, H R (1990) New developments in biochemical mass spectrometry: electrospray ionisation Anal Chem 62, 882 –899 Mann, M and Wilm, M (1995) Electrospray mass spectrometry for protein characterization Trends Biochem Sci 20, 219 –224 Allen, G (1989) Sequencing of proteins and peptides Laboratory techniques in Biochemistry and Molecular Biology (Burdon, R H and van Knippenberg, eds.), Elsevier, Amsterdam 10 Yost, R A and Boyd, R K (1990) Tandem mass spectrometry: quadrupole and hybrid instruments Methods Enzymol 193, 154 – 200 11 Cowley, D J., O’Kane, E., and Todd, R S J (1991) Triazinylaniline derivatives as fluorescence probes Part Absorption and fluorescence in organic solvents and in aqueous media in relation to twisted intramolecular charge-transfer state formation, H bonding and protic equilibria J Chem Soc Perkin Trans 2, 1495–1500 12 Török, K and Trentham, D R (1994) Mechanism of 2-chloro-(ε-amino-Lys75)(6-(4-N,N-diethylamino-phenyl)-1,3,5-triazin-4-yl)-calmodulin interactions with smooth muscle myosin light chain kinase and derived peptides Biochemistry 33, 12,807–12,820 13 Török, K., Cowley, D J., Brandmeier, B D., Howell, S., Aitken A., and Trentham D R (1998) Inhibition of calmodulin-activated smooth muscle myosin light chain Spatial Distribution of Ca2+-Binding Proteins 14 15 16 17 407 kinase by calmodulin binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives Biochemistry 37, 6188 –6198 Teruel, M N and Meyer, T (1997) Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells Biophys J 73, 1785 – 1796 Wilding, M., Török K., and Whitaker M J (1995) Activation-dependent and activation-independent localisation of calmodulin to the mitotic apparatus during the first cell cycle of the Lytenichus pictus embryo Zygote 3, 219 –224 Pawley, J., ed (1989) The Handbook of Biological Confocal Microscopy IMR Press, Madison, Wisconsin Török, K., Wilding, M., Groigno, L., Patel, R D., and Whitaker, M J (1998) Spatial dynamics of calmodulin activation during mitosis in early sea urchin embryos Curr Biol 8, 692 –699 408 Török et al Index 409 Index A Absorption spectroscopy, 43, 46, 52 Aggregation, see Light Scattering; Sedimentation equilibrium Agonists, see Calmodulin, agonists Amlexanox, see Calmodulin, agonists Analytical ultracentrifugation, see Sedimentation equilibrium Annexins, calcium-binding sequences, 231– 232, 246, see also Multiple sequence alignment B β-Galactosidase assay, 356, 357– 358, 359 – 360, 361 BAPTA, 372, see also Calcium, indicator dyes C 13C, see NMR, isotope labeling C2 domain proteins, calcium-binding sequence, 231– 233, 244, see also Multiple sequence alignment Cadmium, see NMR, cadmium-113 Calbindin, see also EF-hand proteins cadmium-binding, 207 calcium-binding, 163 –164 conformational changes, 164 Calcineurin, see Calmodulin, assays, calcineurin 339 –341, 344 –346, 350 –352 Calcium-binding peptides, synthetic, see EF-hand proteins, synthetic Calcium, binding to proteins, binding constant determination (direct), 5, 11–12, 90 – 93, 95 – 98, 100, 121–123, 223, 370 –373 binding constant determination (indirect), 18 –20, 93 – 94, 98 –100, 222 – 223 computer data fitting, 18 – 20, 33 detection by competitive chelators, 15 – 23, 372 detection by flow dialysis (45Ca), –13 detection by NMR, see NMR, calcium-43 pKa of binding site, 223 – 224 regulation of protein interactions, 106 –109 stoichiometry determination, 20–21, 26 – 33, 162 –163, 380 thermodynamics, see Calorimetry, ITC chelation and decontamination, 15 –17, 21– 22, 53, 97– 98, 170, 213, 372 indicator dyes, see Fluorescence, calcium-binding dyes solution preparation, 97– 98 409 410 substitutes, see Fluorescence, terbium; Gadolinium; Manganese; NMR spectroscopy, cadmium113; NMR spectroscopy, lead-207; Vanadyl Calcium/calmodulindependent kinase II, see Calmodulin, assays, CaMKII Calmodulin, see also EF-hand proteins agonists, 325 – 326, 332–334 assays, 339 – 341, 350 – 352 cadmium-binding, 207 calcineurin 339 – 341, 344 –346, 350 – 352 calcium-binding, 10, 25 – 26, 33 – 38, 90 – 96, 162 CaMKII, 340, 343, 349 – 352, 353 changes in, 37 characterization by mass spec/ HPLC, 388 – 392 cooperativity, 34 – 35 fragments of, 183 –191, 210 – 211 free intracellular levels, 365 – 366, 376 – 379 FTIR studies, 62, 69 –70 fluorescence studies, 77–78, 90 – 96 indicator proteins, see Fluorescent CaM indicator proteins lead-binding, 207, MLCK, 340, 342, 349, 350 – 352, 353, see also MLCK NOS, 339 – 342, 346 – 349, 350 – 353 PDE, 339 – 341, 343 – 344, 350 – 352 soybean, 339 Index spatial cellular distribution, 383, delivery, 393 – 398 localization and activation, 398 – 402 FL-calmodulin, 384, 385 – 393, 402, see also Fluorescein dichlorotriazine (5-DTAF) structure, 148 –152 target-binding, 69 –70, 77–78, 148 –153, 167–170 Calorimetry, DSC, 113 –115 instrumentation, 115, 116 thermodynamic parameters, 117–118 ITC, 121–123 instrumentation, 123 thermodynamic parameters, 125 –126 CaMKII, see Calmodulin, assays, CaMKII, Chelex, see Calcium, chelation and decontamination Chromotography, see also HPLC; Protein purification calmodulin/S100-agonist affinity, 325 – 326, 329 – 336 matrix coupling, 326 – 329 metal chelation, 370 Circular dichroism spectroscopy, 43, 44 buffers, 52 far-UV, 50 – 51 instrumentation, 45 – 47 near-UV, 49 – 50 protein secondary structure, 51– 52 units, 49 Index Citrulline assay, see Calmodulin, assays, NOS Cleavage of proteins, see Proteases Confocal imaging, see Fluorescence, imaging Cromolyn, see Calmodulin, agonists Cyclic nucleotide 3':5'-phosphodiesterase, see Calmodulin, assays, PDE Cytochrome c reduction assay, see Calmodulin, assays, NOS D 2D, see NMR, isotope labeling DG, of unfolding, 117 DDG , of calcium-binding, 23 DANSYL, see Fluorescence, FRET Differential scanning calorimetry, see Calorimetry, DSC Dipolar couplings, see NMR, dipolar couplings Dynamics, see NMR, backbone relaxation E EDTA, see Calcium, chelation and decontamination EF-hand proteins, calcium-binding sequence, 231– 233, 242 – 244, see also Multiple sequence alignment conformational changes on calcium-binding, see Vector geometry mapping fragments of, 183 –185 interhelical angles, see Vector geometry mapping synthetic, 175 –176 EGF domains, 285 – 286 411 backbone relaxation, 290 – 296 structure, 301– 303, 310 EGTA, see Calcium, chelation and decontamination Electron paramagnetic resonance, see ESR Electron spin resonance, see ESR Electroporation, 355 – 357, 358 – 359, 360–361, 385, 393–397, see also β-Galactosidase, Luciferase Enthalpy or protein unfolding, 113, 117 Epidermial growth factor domains, see EGF domains EPR, see ESR ESR, 195 – 203 Eukaryotic protein expression, 373 – 374 Evolutionary relationships, see Multiple sequence alignments, phylogentic analysis F Fluorescein dichlorotriazine (5-DTAF), 384, 385 – 388 Fluorescence, analysis of calcium-binding proteins, 83 – 85, 89 – 90, 95 – 98 Ca2+ on-rates, 95, 100 calcium-binding dyes, 15 –17, 90, 97– 98, 99 –100, 372 dissociation constants, 92 – 94, 98 –100 FRET, 365 – 366 imaging, 398 – 402 inner-filter effects, 96 instrumentation, 79 – 80, 81, 83 scattering effects, 97 412 Index Stern-Volmer plot, 78, 81– 83 terbium, 84 – 85, 101 tryptophan and tyrosine, 75 –79 Flow dialysis, – 5, –11 Fluorescence resonance energy transfer, see Fluorescence, FRET Fluorescent CaM indicator proteins, 365 – 366, 376 – 379 bacterial expression and purification, 367– 370 eukaryotic expression, 373 – 374 quantitation, 374 – 375 Fourier Transform Infrared Spectroscopy, see FTIR spectroscopy Free energy, see DG FTIR spectroscopy, 57–72 data processing, 65 – 69 deuterium shifting, 62 – 63, 71 instrumentation, 57– 60 isotope-edited, 69 –70 time-resolved, 71 Hill coefficient, see Calcium, binding to proteins, stoichiometry determination HPLC, reverse-phase, 177, 179 –180, 330 –332, 384, 386, 388 –392 G Gadolinium, 201 Green Fluorescent Protein, 366, 367–368, 383, see also Fluorescent CaM indicator proteins M Mammalian cells, transfection of; see Electroporation Manganese, 200 Mass spectrometry, electrospray ionization (ESI), 162 –165, 386 –392 Matrilysin, 165 Microinjection, 385, 397– 398 Minimal media, see NMR, isotope labeling MLCK, 150 –153, see also Calmodulin, assays, MLCK calmodulin-binding domain, 148 –150, 167–170 H Heat capacity,117–118 change on binding (DC p ), 121 partial C p (T) of protein, 116 High performance liquid chromotography, see HPLC High pressure liquid chromotography, see HPLC I Infrared Spectroscopy, see FTIR spectroscopy Interhelical angles, see Vector geometry mapping Isothermal scanning calorimetry, see Calorimetry, ISC K, L Kinetics, see Protein-protein interactions, and Calcium, binding to proteins Lead, see NMR, lead-207 Ligand binding curves, see Calcium, binding to proteins Light scattering, 127–131 Luciferase assay, 356, 357– 358, 360 Index Molecular modeling, 147–148, 157, 236 Multiple sequence alignment, 231– 233, 238, 241– 249 algorithms and programs, 235, 237, 246 – 249 analysis, 235 – 237 phylogenetic analysis, 236 – 237, 239 – 241 sequence retrieval, 234 –235, 237–238 substitution matrices, 238 Myosin Light Chain Kinase, see MLCK N 15N, see NMR, isotope labeling 15N relaxation, see NMR, backbone relaxation NADPH oxidation assay, see Calmodulin, assays, NOS Nitric oxide synthase, see Calmodulin, assays, NOS NMR spectroscopy, backbone relaxation, 285 – 293 cadmium-113, 205 – 214 chemical exchange, 208 – 209 calcium-43, 217 – 228 chemical shift anisotropy, see CSA CSA, 213 – 214 diffusion tensor, 286, 293 – 295 dipolar couplings, 301– 303 alignment additives, 304 – 305, 312 analysis, 306 – 308 field effects, 312 structure refinement, 308 – 310 validation, 310 – 312 estimation of tc , 290 – 291 413 exchange contributions, 286–287, 291– 292, 295, 296 – 297 HN correlation (HSQC) type spectra, 307 isotope labeling, 255 – 256 13C/ 15N, 256–259, 260 – 262 2H and 2H/13C/ 15N, 258, 259 – 265 lead-207, 205 – 214 order parameters, 287, 295 quadrupolar relaxation, 218 – 220, 221 structure determination, 267– 279, see also NMR spectroscopy, dipolar couplings ambiguous restraints, 271– 273 calcium restraints, 277– 278 NOE and 3J restraints, 268, 275 – 279 pseudoatom corrections, 276 – 277 structure calculation, 269 – 270, 273 – 275 validation, 270 NOS, see Calmodulin, assays, NOS Nuclear magnetic resonance spectroscopy, see NMR spectroscopy O Order parameters, see NMR, backbone relaxation Oxyhemoglobin assay, see Calmodulin, assays, NOS P Parvalbumin, 211– 212 PDE, see Calmodulin, assays, PDE Peptide synthesis, 176 –177, 178–179 414 Phenothiazines, see Calmodulin, agonists Phosphodiesterase, see Calmodulin, assays, PDE Phylogenetic trees, see Multiple sequence alignment, phylogenetic analysis Proteases, thrombin, 184 –184, 187, 189 –190 trypsin, 184 –189, 384, 388 Protein aggregation, see Light Scattering; Analytical Ultracentrifugation Protein concentration determination, 46, 52, 80, 83, 118, 124, 155 –156 Protein expression, 260, 262–264 antibiotics, 257 Protein folding/unfolding, energies, 113, see also Enthalpy Protein-protein interactions, see also CD spectroscopy, by ESI-MS, 167–168, 172 by SPR, 105 –109 thermodynamics, see Calorimetry, ITC Protein purification, see Chromotography and HPLC Protein structure, primary, see Multiple sequence alignment secondary, 50 – 52, 67–70 prediction, 235 – 236 tertiary, see Molecular modeling; NMR spectroscopy, structure deterimination; Vector geometry mapping Proteolysis, see Proteases Index R Radius of gyration (Rg ), 145 –147 Recoverin, 164 Residual dipolar couplings, see NMR, dipolar couplings S S-100 proteins, see also EFhand proteins purification, 329 – 336 SAXS, see Small-angle X-ray scattering ScaM, see Calmodulin, soybean Scatchard plot, 28 – 29 Secondary structure, see Protein structure, secondary Sedimentation equilibrium, 127, 131–135 Small-angle X-ray scattering, 137–138 data analysis, 145 –148 facilities and instrumentation, 138 –140 theory, 141–143 Soybean calmodulin, see Calmodulin, soybean Spin labeling, paramagnetic, 196 –197, 198 –199 SPR, 103 –104 calcium-dependent interactions, 106 –107 instrumentation, 104 –105 kinetics, 107–109 Surface Plasmon Resonance, see SPR T T1 / T2 relaxation, see NMR spectroscopy, backbone relaxation Index Terbium, see Fluorescence, terbium Thermodynamic parameters, 220, see also Calorimetry Thrombin, see Proteases, thrombin Tranilast, see Calmodulin, agonists Transfection, see Electroporation Transformation, see Bacterial transformation Troponin C, see also EF-hand proteins interaction with Troponin I, 153 –155 415 U–W Ultraviolet spectroscopy, see Absorption spectroscopy UV-Vis Spectroscopy, see Absorption spectroscopy van’t Hoff enthalpy, see Enthalpy of protein unfolding Vanadyl, 201 Vector geometry mapping, 317– 324 W7, see Calmodulin, agonists METHODS IN MOLECULAR BIOLOGY • 173 TM Series Editor: John M Walker Calcium-Binding Protein Protocols Volume II: Methods and Techniques Edited by Hans J Vogel Department of Biological Sciences, University of Calgary, Calgary, AB, Canada Calcium-binding proteins play an important role in a variety of vital biological processes, ranging from blood clotting and signal transduction in cells, to attaching proteins to membranes and serving as an integral source of calcium In Calcium-Binding Protocols—Volume 1: Reviews and Case Studies and Volume 2: Methods and Techniques—Hans Vogel and a panel of leading researchers review the protein chemistry and behavior of this significant protein class, and provide a comprehensive collection of proven experimental techniques for their study both in vitro and in vivo This second volume focuses on cutting-edge experimental techniques for studying the solution structure, stability, dynamics, calcium-binding properties, and biological activity of calcium-binding protein in general In addition to enzymatic assays and more routine spectroscopic and protein chemistry techniques, there are also NMR approaches, thermodynamic analyses, kinetic measurements such as surface plasmon resonance, strategies for amino acid sequence alignments, and fluorescence methods to study the distribution of calcium and calcium-binding proteins in cells The first companion volume, Reviews and Case Histories sets the stage for this volume by introducing the various classes of intra- and extracellular calcium-binding proteins and their mode of action Comprehensive and highly practical, the two volumes of Calcium-Binding Protocols provide experimental and clinical biologists with a host of advanced experimental methods that can be applied successfully to the study of both existing and newly discovered members of this critically important class of proteins FEATURES • All major biophysical and protein methods to study calcium-binding proteins • Detailed discussion of calcium-binding proteins in vitro and in vivo • Methods using fluorescence spectroscopy, NMR, thermodynamic analysis, and kinetic measurements • Many methods also applicable to proteins that not bind to calcium CONTENTS Part III Methods and Techniques to Study Calcium-Binding Proteins Quantitative Analysis of Ca2+-Binding by Flow Dialysis Calcium Binding to Proteins Studied via Competition with Chromophoric Chelators Deconvolution of Calcium-Binding Curves: Facts and Fantasies Absorption and Circular Dichroism Spectroscopy Fourier Transform Infrared Spectroscopy of Calcium-Binding Proteins Steady-State Fluorescence Spectroscopy Fluorescence Methods for Measuring Calcium Affinity and Calcium Exchange with Proteins Surface Plasmon Resonance of CalciumBinding Proteins Differential Scanning Calorimetry Isothermal Titration Calorimetry Multiangle Laser Light Scattering and Sedimentation Equilibrium Small-Angle Solution Scattering Reveals Information on Conformational Dynamics in Calcium-Binding Proteins and in their Interactions with Regulatory Targets Investigation of Calcium-Binding Proteins Using Electrospray Ionization Mass Spectrometry Synthetic Calcium-Binding Peptides Proteolytic Fragments of Calcium-Binding Proteins Electron Magnetic Resonance Studies of Calcium-Binding Proteins Cadmium-113 and Lead-207 NMR Spectroscopic Studies of Calcium-Binding Proteins Calcium-43 of NMR of Calcium-Binding Proteins Exploring Familial Methods in Molecular BiologyTM • 173 CALCIUM-BINDING PROTEIN PROTOCOLS VOLUME II: METHODS AND TECHNIQUES ISBN: 0-89603-689-8 humanapress.com Relationships Using Multiple Sequence Alignment Structure Determination by NMR: Isotope Labeling Protein Structure Calculation from NMR Data Shape and Dynamics of a Calcium-Binding Protein Investigated by Nitrogen-15 NMR Relaxation The Use of Dipolar Couplings for the Structure Refinement of a Pair of Calcium-Binding EGF Domains Vector Geometry Mapping: A Method to Characterize the Conformation of HelixLoop-Helix Calcium-Binding Proteins Use of Calmodulin Antagonists and S-100 Protein Interacting Drugs for Affinity Chromatography Enzymatic Assays to Compare Calmodulin Isoforms, Mutants, and Chimeras Gene Expression in Transfected Cells Monitoring the Intracellular Free Ca2+-Calmodulin Concentration with Genetically-Encoded Fluorescent Indicator Proteins Studying the Spatial Distribution of Ca2+-Binding Proteins: How Does it Work for Calmodulin? Index 90000 780896 036895 ... Jürgen Krauss, 20 02 206 Endothelin Protocols, edited by Janet J Maguire and Anthony P Davenport, 20 02 205 E coli Gene Expression Protocols, edited by Peter E Vaillancourt, 20 02 204 Molecular Cytogenetics:... and Protocols, edited by Barry W Hicks, 20 02 1 82 In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Braman, 20 02 181 Genomic Imprinting: Methods and Protocols, edited by Andrew Ward, 20 02. .. Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J Vogel, 20 01 1 72 Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J Vogel, 20 01