Eugene A. Permyakov received his PhD in physics and mathematics at the Moscow Institute of Physics and Technology in 1976 and defended his Doctor of Science dissertation in biology at Moscow State University in 1989. From 1970 to 1994, he worked at the Institute of Theoretical and Experimental Bio-physics of the Russian Academy of Sciences. Since 1994 he has been the Director of the Institute for Biological Instrumentation of the Russian Academy of Sciences. He is known for his work on metal binding proteins and the intrinsic fluorescence method. His primary research focus is the study of physico-chemical and functional properties of metal binding (especially calcium-binding) proteins. Robert H. Kretsinger received his PhD in biophysics at the Massachusetts Institute of Technology under the supervision of Alex Rich in 1964. In 1967 he set up a protein crystallography lab in the Department of Biology at the University of Virginia, where he¿presently teaches, and determined the crystal structure of parvalbumin as well as describing the EF-hand domain in 1971. He has subsequently investigated the structures, functions, and evolution of various EF-hand homologs, along with other protein families. From 1978 to 1995, he directed a national facility to build multiwire area detectors for x-ray diffraction and to collect data from protein crystals.

• Preface xi1 Historical Perspective 11.1 Biomineralization 11.2 Coagulation 41.3 Secondary Messengers (Anticipated) 61.4 Colloids 61.5 Cross-Linking and Cell Surfaces 81.6 Secondary Messengers (Updated) 101.7 Pumps Channels and Ionophores 121.8 Calcium Binding Proteins 11.9 Secondary Messengers (Yet Again) 141.10 Mitochondria 161.11 Pumps Channels and Ionophores 181.12 Hormones 231.13 Measurement 261.14 Biomineralization: Redux 292 Physiological Processes Involving Calcium Binding Proteins 332.1 Calcium as a Secondary Messenger 342.2 Calcium Buffers 412.3 Calcium Pumps and Channels 412.4 Mitochondria 422.5 Eubacteria 432.6 Calcium and Extracellular Proteins 452.7 Biomineralization 462.8 Calcium and Viruses 513 Comparison of the Ca 2+ Ion with Other Metal Cations 553.1 Calcium Isotopes 553.2 Calcium in the Environment 553.3 Uses of Calcium 563.4 Health Effects of Calcium 573.5 Biologically Significant Metals in the Periodic Table 573.6 Hydration of Metal Ions 593.7 “Hard” and “Soft” Metal Ions 604 Complexes of Calcium and Other Cations with Compounds of Low Molecular Weight 634.1 Crystal Structures of Complexes of Calcium with Low Molecular Weight Compounds 634.2 Dissociation Constants of Calcium and Analogs with Small Compounds 664.3 Solubilities of Calcium and Analogs with Small Compounds 675 Stoichiometry Kinetics and Thermodynamics of Calcium Binding 715.1 Stoichiometry Affinity and Cooperativity of Binding 715.2 Kinetics of Binding 775.3 Partition of Free Energy of Binding (�G) Among Enthalpy (�H) and Entropy (�S) 786 Experimental Methods Used to Study Calcium Binding to Proteins 836.1 Radioactivity 836.2 Ion-Selective Electrodes 846.3 Calcium Buffers 856.4 Dialysis Equilibrium and Flow 866.5 Proteolysis 886.6 Deuterium Exchange 886.7 Isothermal Titration Calorimetry 896.8 Differential Scanning Calorimetry 916.9 Mass Spectroscopy 926.10 Calcium-Specific Dyes and Fluors 946.11 Atomic Flame Absorption Spectroscopy 976.12 Absorption Spectroscopy 996.13 Fluorescence Spectroscopy 1026.14 Circular Dichroic and Optical Rotatory Dispersion Spectroscopy 1056.15 Nuclear Magnetic Resonance 1076.16 Electron Spin Resonance 1106.17 Surface Plasmon Resonance 1116.18 Extended X-ray Absorption Spectroscopy 1126.19 Small Angle X-ray Scattering 1136.20 Crystallography 1137 Structure and Evolution of Proteins 1177.1 Domain 1177.2 Structure 1197.3 Evolution 1228 Protein Complexes with Metals Other than Calcium 1278.1 Essential Hard Cations 1298.2 Essential Metals with Several Valence States 1528.3 Conclusions 1879 Nonessential Metals 1899.1 Alkali Metals (Group Ia) 1899.2 Alkali Earth Metals (Group IIa) 1939.3 Group IIIa 1979.4 Group IVa 2049.5 Group Va 2069.6 Group VIIa 2069.7 Group VIII 2079.8 Group Ib 2099.9 Mercury (Group IIb) 2109.10 Group IIIb 2119.11 Group IVb 2159.12 Group Vb 2179.13 Polonium (Group VIb) 2199.14 Conclusions and Generalizations 21910 Parvalbumin 22110.1 Structure 22110.2 Function 23111 EF-Hand Proteins 23711.1 CTER (Calmodulin Troponin C Essential and Regulatory Light Chain) Subfamily 23711.2 CPR (Calcineurin B p22 Recoverin) Subfamily 26611.3 S100 Subfamily 27811.4 Penta-EF-Hand Subfamily 29411.5 Proteins with Six EF-Hands 30011.6 Proteins with Eight and 12 EF-Hands 30511.7 Proteins with Four EF-Hands 30611.8 Proteins with Two EF-Hands 31511.9 EF-Hand Proteins in Bacteria and Viruses 32312 Cytosolic Calcium Binding Proteins Lacking EF-Hands 32712.1 Annexins 32812.2 C2-Domain Proteins 33412.3 Calcium ATPases 35012.4 Calcium Binding Proteins of the Endoplasmic Reticulum 35413 Extracellular Calcium Binding Proteins 36113.1 α-Lactalbumin 36213.2 Cell Matrix Proteins 37113.3 Blood-Clotting Proteins 38013.4 Osteocalcin 38313.5 Calcium Binding Lectins 38513.6 Calcium Binding Hydrolytic Enzymes 38913.7 Miscellaneous 39614 Interactions of Calcium Binding Proteins with Other Metal Ions 39914.1 Magnesium 39914.2 Sodium and Potassium 40214.3 Zinc 40314.4 Trace Metals 40615 Interactions of Calcium Binding Proteins with Other Proteins and with Membranes 41115.1 Interactions with Other Proteins 41115.2 Interactions with Membranes 42816 Genetic Engineering of Calcium Binding Proteins 43916.1 Problems 44016.2 Insertion of Reporter Groups 44116.3 Mutations in Calcium Binding Sites 44216.4 Mutations Elsewhere Than in Calcium Binding Sites 44816.5 Generation of Chimeric Proteins 45116.6 Creation of Calcium Binding Sites in Proteins 45316.7 Studies of Protein–Protein and Protein–Membrane Interactions 455References 459Index 567

Eugene A. Permyakov received his PhD in physics and mathematics at the Moscow Institute of Physics and Technology in 1976 and defended his Doctor of Science dissertation in biology at Moscow State University in 1989. From 1970 to 1994, he worked at the Institute of Theoretical and Experimental Bio-physics of the Russian Academy of Sciences. Since 1994 he has been the Director of the Institute for Biological Instrumentation of the Russian Academy of Sciences. He is known for his work on metal binding proteins and the intrinsic fluorescence method. His primary research focus is the study of physico-chemical and functional properties of metal binding (especially calcium-binding) proteins. Robert H. Kretsinger received his PhD in biophysics at the Massachusetts Institute of Technology under the supervision of Alex Rich in 1964. In 1967 he set up a protein crystallography lab in the Department of Biology at the University of Virginia, where he¿presently teaches, and determined the crystal structure of parvalbumin as well as describing the EF-hand domain in 1971. He has subsequently investigated the structures, functions, and evolution of various EF-hand homologs, along with other protein families. From 1978 to 1995, he directed a national facility to build multiwire area detectors for x-ray diffraction and to collect data from protein crystals.

• Preface xi1 Historical Perspective 11.1 Biomineralization 11.2 Coagulation 41.3 Secondary Messengers (Anticipated) 61.4 Colloids 61.5 Cross-Linking and Cell Surfaces 81.6 Secondary Messengers (Updated) 101.7 Pumps Channels and Ionophores 121.8 Calcium Binding Proteins 11.9 Secondary Messengers (Yet Again) 141.10 Mitochondria 161.11 Pumps Channels and Ionophores 181.12 Hormones 231.13 Measurement 261.14 Biomineralization: Redux 292 Physiological Processes Involving Calcium Binding Proteins 332.1 Calcium as a Secondary Messenger 342.2 Calcium Buffers 412.3 Calcium Pumps and Channels 412.4 Mitochondria 422.5 Eubacteria 432.6 Calcium and Extracellular Proteins 452.7 Biomineralization 462.8 Calcium and Viruses 513 Comparison of the Ca 2+ Ion with Other Metal Cations 553.1 Calcium Isotopes 553.2 Calcium in the Environment 553.3 Uses of Calcium 563.4 Health Effects of Calcium 573.5 Biologically Significant Metals in the Periodic Table 573.6 Hydration of Metal Ions 593.7 “Hard” and “Soft” Metal Ions 604 Complexes of Calcium and Other Cations with Compounds of Low Molecular Weight 634.1 Crystal Structures of Complexes of Calcium with Low Molecular Weight Compounds 634.2 Dissociation Constants of Calcium and Analogs with Small Compounds 664.3 Solubilities of Calcium and Analogs with Small Compounds 675 Stoichiometry Kinetics and Thermodynamics of Calcium Binding 715.1 Stoichiometry Affinity and Cooperativity of Binding 715.2 Kinetics of Binding 775.3 Partition of Free Energy of Binding (�G) Among Enthalpy (�H) and Entropy (�S) 786 Experimental Methods Used to Study Calcium Binding to Proteins 836.1 Radioactivity 836.2 Ion-Selective Electrodes 846.3 Calcium Buffers 856.4 Dialysis Equilibrium and Flow 866.5 Proteolysis 886.6 Deuterium Exchange 886.7 Isothermal Titration Calorimetry 896.8 Differential Scanning Calorimetry 916.9 Mass Spectroscopy 926.10 Calcium-Specific Dyes and Fluors 946.11 Atomic Flame Absorption Spectroscopy 976.12 Absorption Spectroscopy 996.13 Fluorescence Spectroscopy 1026.14 Circular Dichroic and Optical Rotatory Dispersion Spectroscopy 1056.15 Nuclear Magnetic Resonance 1076.16 Electron Spin Resonance 1106.17 Surface Plasmon Resonance 1116.18 Extended X-ray Absorption Spectroscopy 1126.19 Small Angle X-ray Scattering 1136.20 Crystallography 1137 Structure and Evolution of Proteins 1177.1 Domain 1177.2 Structure 1197.3 Evolution 1228 Protein Complexes with Metals Other than Calcium 1278.1 Essential Hard Cations 1298.2 Essential Metals with Several Valence States 1528.3 Conclusions 1879 Nonessential Metals 1899.1 Alkali Metals (Group Ia) 1899.2 Alkali Earth Metals (Group IIa) 1939.3 Group IIIa 1979.4 Group IVa 2049.5 Group Va 2069.6 Group VIIa 2069.7 Group VIII 2079.8 Group Ib 2099.9 Mercury (Group IIb) 2109.10 Group IIIb 2119.11 Group IVb 2159.12 Group Vb 2179.13 Polonium (Group VIb) 2199.14 Conclusions and Generalizations 21910 Parvalbumin 22110.1 Structure 22110.2 Function 23111 EF-Hand Proteins 23711.1 CTER (Calmodulin Troponin C Essential and Regulatory Light Chain) Subfamily 23711.2 CPR (Calcineurin B p22 Recoverin) Subfamily 26611.3 S100 Subfamily 27811.4 Penta-EF-Hand Subfamily 29411.5 Proteins with Six EF-Hands 30011.6 Proteins with Eight and 12 EF-Hands 30511.7 Proteins with Four EF-Hands 30611.8 Proteins with Two EF-Hands 31511.9 EF-Hand Proteins in Bacteria and Viruses 32312 Cytosolic Calcium Binding Proteins Lacking EF-Hands 32712.1 Annexins 32812.2 C2-Domain Proteins 33412.3 Calcium ATPases 35012.4 Calcium Binding Proteins of the Endoplasmic Reticulum 35413 Extracellular Calcium Binding Proteins 36113.1 α-Lactalbumin 36213.2 Cell Matrix Proteins 37113.3 Blood-Clotting Proteins 38013.4 Osteocalcin 38313.5 Calcium Binding Lectins 38513.6 Calcium Binding Hydrolytic Enzymes 38913.7 Miscellaneous 39614 Interactions of Calcium Binding Proteins with Other Metal Ions 39914.1 Magnesium 39914.2 Sodium and Potassium 40214.3 Zinc 40314.4 Trace Metals 40615 Interactions of Calcium Binding Proteins with Other Proteins and with Membranes 41115.1 Interactions with Other Proteins 41115.2 Interactions with Membranes 42816 Genetic Engineering of Calcium Binding Proteins 43916.1 Problems 44016.2 Insertion of Reporter Groups 44116.3 Mutations in Calcium Binding Sites 44216.4 Mutations Elsewhere Than in Calcium Binding Sites 44816.5 Generation of Chimeric Proteins 45116.6 Creation of Calcium Binding Sites in Proteins 45316.7 Studies of Protein–Protein and Protein–Membrane Interactions 455References 459Index 567

Eugene A. Permyakov received his PhD in physics and mathematics at the Moscow Institute of Physics and Technology in 1976 and defended his Doctor of Science dissertation in biology at Moscow State University in 1989. From 1970 to 1994, he worked at the Institute of Theoretical and Experimental Bio-physics of the Russian Academy of Sciences. Since 1994 he has been the Director of the Institute for Biological Instrumentation of the Russian Academy of Sciences. He is known for his work on metal binding proteins and the intrinsic fluorescence method. His primary research focus is the study of physico-chemical and functional properties of metal binding (especially calcium-binding) proteins. Robert H. Kretsinger received his PhD in biophysics at the Massachusetts Institute of Technology under the supervision of Alex Rich in 1964. In 1967 he set up a protein crystallography lab in the Department of Biology at the University of Virginia, where he¿presently teaches, and determined the crystal structure of parvalbumin as well as describing the EF-hand domain in 1971. He has subsequently investigated the structures, functions, and evolution of various EF-hand homologs, along with other protein families. From 1978 to 1995, he directed a national facility to build multiwire area detectors for x-ray diffraction and to collect data from protein crystals.

• Preface xi1 Historical Perspective 11.1 Biomineralization 11.2 Coagulation 41.3 Secondary Messengers (Anticipated) 61.4 Colloids 61.5 Cross-Linking and Cell Surfaces 81.6 Secondary Messengers (Updated) 101.7 Pumps Channels and Ionophores 121.8 Calcium Binding Proteins 11.9 Secondary Messengers (Yet Again) 141.10 Mitochondria 161.11 Pumps Channels and Ionophores 181.12 Hormones 231.13 Measurement 261.14 Biomineralization: Redux 292 Physiological Processes Involving Calcium Binding Proteins 332.1 Calcium as a Secondary Messenger 342.2 Calcium Buffers 412.3 Calcium Pumps and Channels 412.4 Mitochondria 422.5 Eubacteria 432.6 Calcium and Extracellular Proteins 452.7 Biomineralization 462.8 Calcium and Viruses 513 Comparison of the Ca 2+ Ion with Other Metal Cations 553.1 Calcium Isotopes 553.2 Calcium in the Environment 553.3 Uses of Calcium 563.4 Health Effects of Calcium 573.5 Biologically Significant Metals in the Periodic Table 573.6 Hydration of Metal Ions 593.7 “Hard” and “Soft” Metal Ions 604 Complexes of Calcium and Other Cations with Compounds of Low Molecular Weight 634.1 Crystal Structures of Complexes of Calcium with Low Molecular Weight Compounds 634.2 Dissociation Constants of Calcium and Analogs with Small Compounds 664.3 Solubilities of Calcium and Analogs with Small Compounds 675 Stoichiometry Kinetics and Thermodynamics of Calcium Binding 715.1 Stoichiometry Affinity and Cooperativity of Binding 715.2 Kinetics of Binding 775.3 Partition of Free Energy of Binding (�G) Among Enthalpy (�H) and Entropy (�S) 786 Experimental Methods Used to Study Calcium Binding to Proteins 836.1 Radioactivity 836.2 Ion-Selective Electrodes 846.3 Calcium Buffers 856.4 Dialysis Equilibrium and Flow 866.5 Proteolysis 886.6 Deuterium Exchange 886.7 Isothermal Titration Calorimetry 896.8 Differential Scanning Calorimetry 916.9 Mass Spectroscopy 926.10 Calcium-Specific Dyes and Fluors 946.11 Atomic Flame Absorption Spectroscopy 976.12 Absorption Spectroscopy 996.13 Fluorescence Spectroscopy 1026.14 Circular Dichroic and Optical Rotatory Dispersion Spectroscopy 1056.15 Nuclear Magnetic Resonance 1076.16 Electron Spin Resonance 1106.17 Surface Plasmon Resonance 1116.18 Extended X-ray Absorption Spectroscopy 1126.19 Small Angle X-ray Scattering 1136.20 Crystallography 1137 Structure and Evolution of Proteins 1177.1 Domain 1177.2 Structure 1197.3 Evolution 1228 Protein Complexes with Metals Other than Calcium 1278.1 Essential Hard Cations 1298.2 Essential Metals with Several Valence States 1528.3 Conclusions 1879 Nonessential Metals 1899.1 Alkali Metals (Group Ia) 1899.2 Alkali Earth Metals (Group IIa) 1939.3 Group IIIa 1979.4 Group IVa 2049.5 Group Va 2069.6 Group VIIa 2069.7 Group VIII 2079.8 Group Ib 2099.9 Mercury (Group IIb) 2109.10 Group IIIb 2119.11 Group IVb 2159.12 Group Vb 2179.13 Polonium (Group VIb) 2199.14 Conclusions and Generalizations 21910 Parvalbumin 22110.1 Structure 22110.2 Function 23111 EF-Hand Proteins 23711.1 CTER (Calmodulin Troponin C Essential and Regulatory Light Chain) Subfamily 23711.2 CPR (Calcineurin B p22 Recoverin) Subfamily 26611.3 S100 Subfamily 27811.4 Penta-EF-Hand Subfamily 29411.5 Proteins with Six EF-Hands 30011.6 Proteins with Eight and 12 EF-Hands 30511.7 Proteins with Four EF-Hands 30611.8 Proteins with Two EF-Hands 31511.9 EF-Hand Proteins in Bacteria and Viruses 32312 Cytosolic Calcium Binding Proteins Lacking EF-Hands 32712.1 Annexins 32812.2 C2-Domain Proteins 33412.3 Calcium ATPases 35012.4 Calcium Binding Proteins of the Endoplasmic Reticulum 35413 Extracellular Calcium Binding Proteins 36113.1 α-Lactalbumin 36213.2 Cell Matrix Proteins 37113.3 Blood-Clotting Proteins 38013.4 Osteocalcin 38313.5 Calcium Binding Lectins 38513.6 Calcium Binding Hydrolytic Enzymes 38913.7 Miscellaneous 39614 Interactions of Calcium Binding Proteins with Other Metal Ions 39914.1 Magnesium 39914.2 Sodium and Potassium 40214.3 Zinc 40314.4 Trace Metals 40615 Interactions of Calcium Binding Proteins with Other Proteins and with Membranes 41115.1 Interactions with Other Proteins 41115.2 Interactions with Membranes 42816 Genetic Engineering of Calcium Binding Proteins 43916.1 Problems 44016.2 Insertion of Reporter Groups 44116.3 Mutations in Calcium Binding Sites 44216.4 Mutations Elsewhere Than in Calcium Binding Sites 44816.5 Generation of Chimeric Proteins 45116.6 Creation of Calcium Binding Sites in Proteins 45316.7 Studies of Protein–Protein and Protein–Membrane Interactions 455References 459Index 567