Functional Consequences of Protein Dynamics

[摘要] This thesis explores the possible uses of dynamical fluctuations in protein structure for ligand binding and catalysis. Particular emphasis is placed on the dynamic interpretation of allosteric interactions and a statistical mechanical model of dynamic allostery is presented. This model shows that changes in either low frequency collective motions or random uncorrelated atomic fluctuations of the protein induced by the binding of a ligand can alter the binding properties of other remote ligand binding sites giving rise to allostery. Increases in the frequency of vibrational modes and reductions in uncorrelated motions gives rise to positive cooperativity and is equivalent to a stiffening of the protein structure. Small changes in the dynamics can be treated classically with many changes being required to give observed cooperative free energies. Large shifts in low frequency vibrational modes give much larger contributions to the cooperativity and the use of quantum mechanics is required. The dynamic allostery model predicts that cooperativity arising from dynamic changes is predominantly entropic in origin and complements the more conventional models of allostery which invoke changes in the static conformation of the protein involving domain movement, bond rearrangements and electrostatic effects with consequent effects on the enthalpy. The predictions of this model are tested using laser Raman spectroscopy of solid samples to study low frequency modes in proteins and an allosteric model compound. The small organic molecule which displays positive cooperativity between its two binding sites, shows sizeable shifts to higher frequencies in the low frequency spectrum in agreement with the model. The vibrational shifts seen require only the classical version of the model which when combined with changes in the uncorrelated motions of the atoms in the molecule can account for the observed cooperative free energy. The cooperativity is solely entropic in origin in agreement with published results. High frequency spectra of the molecule in various states of ligation are presented and analysed in terms of localised vibrations of atoms and groups of atoms. The low frequency Raman spectra of lysozyme and its complex with the small inhibitor tri-N-acetyl glucosamine, and of trypsin and its complex with pancreatic trypsin inhibitor all displayed a broad band at 20cm. This band is a superposition of a large number of low frequency modes of the protein and the expected shift in frequency of some modes on inhibitor binding is not visible within such a broad band. The allosteric enzyme glyceraldehyde 3-phosphate dehydrogenase and its complex with the cofactor NAD also shows no changes in its low frequency spectrum. These results and their implications are discussed. High frequency Raman spectra of these enzymes are also presented and analysed.

[发布机构] University:University of Glasgow

[关键词] Molecular biology