Real-time NMR of the transient states of proteins
The work described in this thesis is concerned with the development and application of real-time photo-CIDNP (Chemically Induced Dynamic Nuclear Polarisation) to the study of protein structure and folding. Chapters 1 and 2 introduce the protein folding problem, and its study by NMR, then go on to elucidate the mechanisms behind the photo-CIDNP phenomenon. Chapter 3 applies photo-CIDNP spectroscopy to the study of a small cytochrome protein. The difficulties of performing these experiments on chromophore-containing proteins are discussed. Chapter 4 begins with the development of a rapid mixing device for use in real-time NMR and CIDNP studies. Experiments used to characterise the device are presented. This chapter then goes on to describe CIDNP pulse labelling experiments, used to investigate the surface structure of some molten globule states of two a-lactalbumins. This chapter concludes with an application of the rapid mixing device to the real-time refolding of hen egg white lysozyme. Chapter 5 extends the work of the previous chapter, studying the real-time refolding of bovine pancreatic ribonuclease A. Refolding studies are performed from different denaturing conditions, and the effects of sample heating during the real-time CIDNP experiment are discussed. Chapter 6 describes the use of illumination during an NMR experiment to study the conformational changes in a plant blue light receptor protein, phototropin. The structural changes are characterised with 2-dimensional NMR spectroscopy and photo-CIDNP. The kinetics of the ground state recovery are also investigated by real-time NMR spectroscopy. Chapter 7 uses calculated hyperfine coupling constants and a radical pair diffusion model from the literature to simulate the nuclear polarisation obtained for the amino acid tryptophan. Comparisons are made between theory and experiment. Chapter 8 describes the structural characterisation of a homologous series of de novo peptides, designed for subsequent use in EPR experiments when derivatised with a suitable spin label.