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Microcalorimetry of Macromolecules: The Physical Basis of Biological Structures

ISBN: 978-1-118-33749-3
450 pages
May 2012
Microcalorimetry of Macromolecules: The Physical Basis of Biological Structures (1118337492) cover image

Examining the physical basis of the structure of macromolecules—proteins, nucleic acids, and their complexes—using calorimetric techniques

Many scientists working in biology are unfamiliar with the basics of thermodynamics and its role in determining molecular structures. Yet measuring the heat of structural change a molecule undergoes under various conditions yields information on the energies involved and, thus, on the physical bases of the considered structures. Microcalorimetry of Macromolecules offers protein scientists unique access to this important information.

Divided into thirteen chapters, the book introduces readers to the basics of thermodynamics as it applies to calorimetry, the evolution of the calorimetric technique, as well as how calorimetric techniques are used in the thermodynamic studies of macromolecules, detailing instruments for measuring the heat effects of various processes. Also provided is general information on the structure of biological macromolecules, proteins, and nucleic acids, focusing on the key thermodynamic problems relating to their structure. The book covers:

  • The use of supersensitive calorimetric instruments, including micro and nano-calorimeters for measuring the heat of isothermal reactions (Isothermal Titration Nano-Calorimeter), the heat capacities over a broad temperature range (Scanning Nano-Calorimeter), and pressure effects (Pressure Perturbation Nano-Calorimeter)
  • Two of the simplest but key structural elements: the α and polyproline helices and their complexes, the α-helical coiled-coil, and the pyroline coiled-coils
  • Complicated macromolecular formations, including small globular proteins, multidomain proteins and their complexes, and nucleic acids
  • Numerous examples of measuring the ground state of protein energetics, as well as changes seen when proteins interact

The book also reveals how intertwined structure and thermodynamics are in terms of a macromolecule's organization, mechanism of formation, the stabilization of its three-dimensional structure, and ultimately, its function. The first book to describe microcalorimetric technique in detail, enough for graduate students and research scientists to successfully plumb the structural mysteries of proteins and the double helix, Microcalorimetry of Macromolecules is an essential introduction to using a microcalorimeter in biological studies.

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1 Introduction 1

2 Methodology 5

2.1 Thermodynamic Basics of Calorimetry, 5

2.1.1 Energy, 5

2.1.2 Enthalpy, 6

2.1.3 Temperature, 6

2.1.4 Energy Units, 7

2.1.5 Heat Capacity, 8

2.1.6 Kirchhoff’s Relation, 9

2.1.7 Entropy, 11

2.1.8 Gibbs Free Energy, 13

2.2 Equilibrium Analysis, 13

2.2.1 Two-State Transition, 13

2.2.2 Derivatives of the Equilibrium Constant, 15

2.3 Aqueous Solutions, 16

2.3.1 Specifi city of Water as a Solvent, 16

2.3.2 Acid–Base Equilibrium, 18

2.3.3 Partial Quantities, 20

2.4 Transfer of Solutes into the Aqueous Phase, 23

2.4.1 Hydration Effects, 23

2.4.2 Hydrophobic Force, 25

2.4.3 Hydration of Polar and Nonpolar Groups, 28

References, 32

3 Calorimetry 33

3.1 Isothermal Reaction Microcalorimetry, 33

3.1.1 The Heat of Mixing Reaction, 33

3.1.2 Mixing of Reagents in Comparable Volumes, 35

3.1.3 Isothermal Titration Microcalorimeter, 36

3.1.4 ITC Experiments, 38

3.1.5 Analysis of the ITC Data, 41

3.2 Heat Capacity Calorimetry, 43

3.2.1 Technical Problems, 43

3.2.2 Differential Scanning Microcalorimeter, 44

3.2.3 Determination of the Partial Heat Capacity of Solute Molecules, 53

3.2.4 DSC Experiments, 55

3.2.5 Determination of the Enthalpy of a Temperature-Induced Process, 56

3.2.6 Determination of the van’t Hoff Enthalpy, 58

3.2.7 Multimolecular Two-State Transition, 59

3.2.8 Analysis of the Complex Heat Capacity Profile, 60

3.2.9 Correction for Components Refolding, 61

3.3 Pressure Perturbation Calorimetry, 63

3.3.1 Heat Effect of Changing Pressure, 63

3.3.2 Pressure Perturbation Experiment, 65

References, 67

4 Macromolecules 69

4.1 Evolution of the Concept, 69

4.2 Proteins, 71

4.2.1 Chemical Structure, 71

4.2.2 Physical Structure, 76

4.2.3 Restrictions on the Conformation of Polypeptide Chains, 81

4.2.4 Regular Conformations of Polypeptide Chain Proteins, 82

4.3 Hierarchy in Protein Structure, 86

4.3.1 Tertiary Structure of Proteins, 86

4.3.2 Quaternary Structure of Proteins, 88

4.4 Nucleic Acids, 89

4.4.1 Chemical Structure, 89

4.4.2 Physical Structure, 91

References, 94

5 The α-Helix and α-Helical Coiled-Coil 95

5.1 The α-Helix, 95

5.1.1 Calorimetric Studies of α-Helix Unfolding–Refolding, 95

5.1.2 Analysis of the Heat Capacity Function, 99

5.2 α-Helical Coiled-Coils, 105

5.2.1 Two-Stranded Coiled-Coils, 105

5.2.2 Three-Stranded Coiled-Coils, 110

5.3 α-Helical Coiled-Coil Proteins, 113

5.3.1 Muscle Proteins, 113

5.3.2 Myosin Rod, 115

5.3.3 Paramyosin, 116

5.3.4 Tropomyosin, 117

5.3.5 Leucine Zipper, 118

5.3.6 Discreteness of the Coiled-Coils, 123

References, 124

6 Polyproline-II Coiled-Coils 127

6.1 Collagens, 127

6.1.1 Collagen Superhelix, 127

6.1.2 Hydrogen Bonds in Collagen, 129

6.1.3 Stability of Collagens, 131

6.1.4 Role of Pyrrolidine Rings in Collagen Stabilization, 133

6.2 Calorimetric Studies of Collagens, 135

6.2.1 Enthalpy and Entropy of Collagen Melting, 135

6.2.2 Correlation between Thermodynamic and Structural Characteristics of Collagens, 138

6.2.3 Role of Water in Maintaining the Collagen Structure, 140

6.3 Thermodynamics of Collagens, 141

6.3.1 Cooperativity of Collagen Unfolding, 141

6.3.2 Factors Responsible for Maintaining the Collagen Coiled-Coil, 143

6.3.3 Flexibility of the Collagen Structure, 145

6.3.4 Biological Aspect of the Collagen Stability Problem, 148

References, 150

7 Globular Proteins 153

7.1 Denaturation of Globular Proteins, 153

7.1.1 Proteins at Extremal Conditions, 153

7.1.2 The Main Problems of Protein Denaturation, 154

7.2 Heat Denaturation of Proteins, 155

7.2.1 DSC Studies of Protein Denaturation upon Heating, 155

7.2.2 Reversibility of Heat Denaturation, 155

7.2.3 Cooperativity of Denaturation, 156

7.2.4 Heat Capacity of the Native and Denatured States, 158

7.2.5 Functions Specifying Protein Stability, 161

7.3 Cold Denaturation, 167

7.3.1 Proteins at Low Temperatures, 167

7.3.2 Experimental Observation of Cold Denaturation, 168

7.4 pH-Induced Protein Denaturation, 173

7.4.1 Isothermal pH Titration of Globular Proteins, 173

7.5 Denaturant-Induced Protein Unfolding, 175

7.5.1 Use of Denaturants for Estimating Protein Stability, 175

7.5.2 Calorimetric Studies of Protein Unfolding by Denaturants, 176

7.5.3 Urea and GuHCl Interactions with Protein, 179

7.6 Unfolded State of Protein, 182

7.6.1 Completeness of Protein Unfolding at Denaturation, 182

7.6.2 Thermodynamic Functions Describing Protein States, 186

References, 190

8 Energetic Basis of Protein Structure 193

8.1 Hydration Effects, 193

8.1.1 Proteins in an Aqueous Environment, 193

8.1.2 Hydration of Protein Groups, 194

8.1.3 Hydration of the Folded and Unfolded Protein, 199

8.2 Protein in Vacuum, 202

8.2.1 Heat Capacity of Globular Proteins, 202

8.2.2 Enthalpy of Protein Unfolding in Vacuum, 204

8.2.3 Entropy of Protein Unfolding in Vacuum, 210

8.3 Back into the Water, 214

8.3.1 Enthalpies of Protein Unfolding in Water, 214

8.3.2 Hydrogen Bonds, 216

8.3.3 Hydrophobic Effect, 218

8.3.4 Balance of Forces Stabilizing and Destabilizing Protein Structure, 219

References, 223

9 Protein Folding 225

9.1 Macrostabilities and Microstabilities of Protein Structure, 225

9.1.1 Macrostability of Proteins, 225

9.1.2 Microstability of Proteins, 226

9.1.3 Packing in Protein Interior, 228

9.2 Protein Folding Technology, 233

9.2.1 Intermediate States in Protein Folding, 233

9.2.2 Molten Globule Concept, 234

9.3 Formation of Protein Structure, 241

9.3.1 Transient State in Protein Folding, 241

9.3.2 Mechanism of Cooperation, 242

9.3.3 Thermodynamic States of Proteins, 243

References, 245

10 Multidomain Proteins 249

10.1 Criterion of Cooperativity, 249

10.1.1 Deviations from a Two-State Unfolding–Refolding, 249

10.1.2 Papain, 250

10.1.3 Pepsinogen, 251

10.2 Proteins with Internal Homology, 255

10.2.1 Evolution of Multidomain Proteins, 255

10.2.2 Ovomucoid, 255

10.2.3 Calcium-Binding Proteins, 258

10.2.4 Plasminogen, 263

10.2.5 Fibrinogen, 264

10.2.6 Fibronectin, 267

10.2.7 Discreteness in Protein Structure, 268

References, 271

11 Macromolecular Complexes 273

11.1 Entropy of Association Reactions, 273

11.1.1 Thermodynamics of Molecular Association, 273

11.1.2 Experimental Verifi cation of the Translational Entropy, 275

11.2 Calorimetry of Association Entropy, 277

11.2.1 SSI Dimer Dissociation, 277

11.2.2 Dissociation of the Coiled-Coil, 283

11.2.3 Entropy Cost of Association, 285

11.3 Thermodynamics of Molecular Recognition, 286

11.3.1 Calorimetry of Protein Complex Formation, 286

11.3.2 Target Peptide Recognition by Calmodulin, 287

11.3.3 Thermodynamic Analysis of Macromolecular Complexes, 293

References, 295

12 Protein–DNA Interaction 297

12.1 Problems, 297

12.1.1 Two Approaches, 297

12.1.2 Protein Binding to the DNA Grooves, 299

12.2 Binding to the Major Groove of DNA, 300

12.2.1 Homeodomains, 300

12.2.2 Binding of the GCN4 bZIP to DNA, 307

12.2.3 Heterodimeric bZIP Interactions with the Asymmetric DNA Site, 313

12.2.4 IRF Transcription Factors, 317

12.2.5 Binding of NF-κB to the PRDII Site, 320

12.3 Binding to the Minor Groove of DNA, 322

12.3.1 AT-Hooks, 322

12.3.2 HMG Boxes, 326

12.4 Comparative Analysis of Protein–DNA Complexes, 331

12.4.1 Sequence-Specifi c versus Non-Sequence-Specifi c HMGs, 331

12.4.2 Salt-Dependent versus Salt-Independent Components of Binding, 336

12.4.3 Minor versus Major Groove Binding, 339

12.5 Concluding Remarks, 345

12.5.1 Assembling Multicomponent Protein–DNA Complex, 345

12.5.2 CC Approach versus PB Theory, 346

References, 347

13 Nucleic Acids 353

13.1 DNA, 353

13.1.1 Problems, 353

13.1.2 Factors Affecting DNA Melting, 354

13.2 Polynucleotides, 357

13.2.1 Melting of Polynucleotides, 357

13.2.2 Calorimetry of Poly(A)·Poly(U), 358

13.3 Short DNA Duplexes, 361

13.3.1 Calorimetry of Short DNA Duplexes, 361

13.3.2 Specifi city of the AT-rich DNA Duplexes, 366

13.3.3 DNA Hydration Studied by Pressure Perturbation Calorimetry, 372

13.3.4 The Cost of DNA Bending, 375

13.4 RNA, 376

13.4.1 Calorimetry of RNA, 376

13.4.2 Calorimetric Studies of Transfer RNAs, 378

References, 383

Index 387

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PETER L. PRIVALOV is a Professor of Biology and Biophysics at the Johns Hopkins University since 1991. He received his PhD in physics from the University of Georgia, Tbilisi (former USSR), and his DrSc in biophysics from the Institute of Biophysics, Russian Academy of Sciences, Moscow. For many years, he headed the Laboratory of Thermodynamics at the Protein Research Institute of the Russian Academy of Sciences. He is the author of 230 scientific papers published in various international journals and periodicals.

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