Proteins

David Whitford, Queen Mary & Westfield College, London, UK.

Preface xi

1 An Introduction to protein structure and function 1

A brief and very selective historical perspective 1

The biological diversity of proteins 5

Proteins and the sequencing of the human and other genomes 9

Why study proteins? 9

2 Amino acids: the building blocks of proteins 13

The 20 amino acids found in proteins 13

The acid–base properties of amino acids 14

Stereochemical representations of amino acids 15

Peptide bonds 16

The chemical and physical properties of amino acids 23

Detection, identification and quantification of amino acids and proteins 32

Stereoisomerism 34

Non-standard amino acids 35

Summary 36

Problems 37

3 The three-dimensional structure of proteins 39

Primary structure or sequence 39

Secondary structure 39

Tertiary structure 50

Quaternary structure 62

The globin family and the role of quaternary structure in modulating activity 66

Immunoglobulins 74

Cyclic proteins 81

Summary 81

Problems 83

4 The structure and function of fibrous proteins 85

The amino acid composition and organization of fibrous proteins 85

Keratins 86

Fibroin 92

Collagen 92

Summary 102

Problems 103

5 The structure and function of membrane proteins 105

The molecular organization of membranes 105

Membrane protein topology and function seen through organization of the erythrocyte membrane 110

Bacteriorhodopsin and the discovery of seven transmembrane helices 114

The structure of the bacterial reaction centre 123

Oxygenic photosynthesis 126

Photosystem I 126

Membrane proteins based on transmembrane β barrels 128

Respiratory complexes 132

Complex III, the ubiquinol-cytochrome c oxidoreductase 132

Complex IV or cytochrome oxidase 138

The structure of ATP synthetase 144

ATPase family 152

Summary 156

Problems 159

6 The diversity of proteins 161

Prebiotic synthesis and the origins of proteins 161

Evolutionary divergence of organisms and its relationship to protein structure and function 163

Protein sequence analysis 165

Protein databases 180

Gene fusion and duplication 181

Secondary structure prediction 181

Genomics and proteomics 183

Summary 187

Problems 187

7 Enzyme kinetics, structure, function, and catalysis 189

Enzyme nomenclature 191

Enzyme co-factors 192

Chemical kinetics 192

The transition state and the action of enzymes 195

The kinetics of enzyme action 197

Catalytic mechanisms 202

Enzyme structure 209

Lysozyme 209

The serine proteases 212

Triose phosphate isomerase 215

Tyrosyl tRNA synthetase 218

EcoRI restriction endonuclease 221

Enzyme inhibition and regulation 224

Irreversible inhibition of enzyme activity 227

Allosteric regulation 231

Covalent modification 237

Isoenzymes or isozymes 241

Summary 242

Problems 244

8 Protein synthesis, processing and turnover 247

Cell cycle 247

The structure of Cdk and its role in the cell cycle 250

Cdk–cyclin complex regulation 252

DNA replication 253

Transcription 254

Eukaryotic transcription factors: variation on a ‘basic’ theme 261

The spliceosome and its role in transcription 265

Translation 266

Transfer RNA (tRNA) 267

The composition of prokaryotic and eukaryotic ribosomes 269

A structural basis for protein synthesis 272

An outline of protein synthesis 273

Antibiotics provide insight into protein synthesis 278

Affinity labelling and RNA ‘footprinting’ 279

Structural studies of the ribosome 279

Post-translational modification of proteins 287

Protein sorting or targeting 293

The nuclear pore assembly 302

Protein turnover 303

Apoptosis 310

Summary 310

Problems 312

9 Protein expression, purification and characterization 313

The isolation and characterization of proteins 313

Recombinant DNA technology and protein expression 313

Purification of proteins 318

Centrifugation 320

Solubility and ‘salting out’ and ‘salting in’ 323

Chromatography 326

Dialysis and ultrafiltration 333

Polyacrylamide gel electrophoresis 333

Mass spectrometry 340

How to purify a protein? 342

Summary 344

Problems 345

10 Physical methods of determining the three-dimensional structure of proteins 347

Introduction 347

The use of electromagnetic radiation 348

X-ray crystallography 349

Nuclear magnetic resonance spectroscopy 360

Cryoelectron microscopy 375

Neutron diffraction 379

Optical spectroscopic techniques 379

Vibrational spectroscopy 387

Raman spectroscopy 389

ESR and ENDOR 390

Summary 392

Problems 393

11 Protein folding in vivo and in vitro 395

Introduction 395

Factors determining the protein fold 395

Factors governing protein stability 403

Folding problem and Levinthal’s paradox 403

Models of protein folding 408

Amide exchange and measurement of protein folding 411

Kinetic barriers to refolding 412

In vivo protein folding 415

Membrane protein folding 422

Protein misfolding and the disease state 426

Summary 435

Problems 437

12 Protein structure and a molecular approach to medicine 439

Introduction 439

Sickle cell anaemia 441

Viruses and their impact on health as seen through structure and function 442

HIV and AIDS 443

The influenza virus 457

p53 and its role in cancer 470

Emphysema and α1-antitrypsin 475

Summary 478

Problems 479

Epilogue 481

Glossary 483

Appendices 491

Bibliography 495

References 499

Index 511