Summary

This thesis describes a preliminary experimental investigation of the use of electron spin resonance (ESR) as a method for studying the conformational changes associated with the folding of proteins and peptides. The study represents the first step towards establishing a new experimental technique. This thesis aims to investigate the feasibility of exploiting ESR for the study of singly- and doubly- spin-labelled peptides and proteins of well-known structure, and to demonstrate under which conditions this new technique might be developed into a superior method for the investigation of sub-millisecond protein folding events.

Chapter 1 introduces the theory behind the project; basic ESR theory is discussed, together with the premise for studying protein folding. Chapter 2 gives the aims of the investigation and a short overview of other possible spectroscopic methods of investigating protein folding such as fluorescence energy transfer. In chapter 3, the techniques used in this project are outlined; these include spin labelling, circular dichroism, ESR instrumentation and the computer simulation program used to interpret the ESR spectra. Preliminary investigations into the labelling and manipulation of a well characterised and studied protein (haemoglobin) and a 14 residue custom made peptide, chosen for its suitability in later experiments, are described in chapter 4. Chapter 5 contains the experimental results and outlines their interpretation. Since dipolar interactions between pairs of spin labels may lead to information about the distance between them, a doubly-labelled peptide is investigated in chapter 5. A discussion as to which peptide should be used in these spin-labelling experiments is also presented. Finally, chapter 6 concludes the work and gives a future perspective on the feasibility of using ESR together with the technique of spin labelling to investigate real time protein folding.