Contents

Abbreviations

Introduction 1

Chapter 1 - Theory 3

1.1 Basic Electron Spin Resonance 3

1.2 The Protein Folding Problem 9

Chapter 2 - The Aims of the Investigation 11

Chapter 3 - Methods 13

3.1 Spin Labels 13

3.2 Circular Dichroism (CD) 16

3.3 The ESR Spectrometer Instrumentation 17

3.4 Simulations 23

Chapter 4 - The Preliminary Experiments and Results 24

4.1 Human Haemoglobin 24

4.2 Haemoglobin Purchased from Sigma 26

4.3 Oxy-Haemoglobin 31

4.4 The Choice of Peptide for Labelling Experiments 39

4.5 Labelling Pep1 and the Experiments 41

Chapter 5 - The Experiments with the Doubly-Labelled Peptide and Corresponding Singly-Labelled Versions 44

5.1 The Peptides Used 44

5.2 Preparing the Peptides 45

5.3 CD and ESR Experiments 46

5.4 Simulations, Interpretations and Discussion 55

Chapter 6 – Conclusions and Future Work 61

References 64

Appendix 1 – Amino Acids and Proteins I

Appendix 2 – The Method for Labelling and Purifying Pep2 IV