Microcalorimetry of Macromolecules

ISBN-13: 9781118104514 / Angielski / Twarda / 2012 / 404 str.

Peter L. Privalov

Opis

This is the first textbook on the microcalorimetry of biological molecules.

Spis treści

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

O autorze

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.

Blurb

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.