The physiological functions of many proteins are associated with conformational changes in their tertiary and quaternary structure. We have developed and employed two methods to investigate possible intermediate states of these transitions; high pressure crystallography is used to "squeeze" the protein into a semi-stable transition state which can then be investigated crystallographically; semi-time resolved cryocrystallography can be employed for slow transformations triggered by external agents.

Rhombohedral insulin crystals were used for these studies as the insulin hexamer has been shown previously to undergo three different, chemically induced transitions.

The first part of this work is concerned with the design and application of the pressure cells used. Extensive experimentation using a brass hydrostatic cell (limited rotation range) indicated time-dependent unit cell volume changes above 5 bar nitrogen gas pressure. These changes were reversible i.e., the unit cell volume returned to its original size after depressurisation. A reordering of the insulin structure was observed at 10 bar; possibly indicative of a conformational change.

Further experiments using a capillary cell failed to repeat these results. However, the new cell permits the recording of complete data sets for structural studies.

Experiments using a uniaxial, mechanical pressure cell proved inconclusive and a very high pressure (1000 bar) beryllium hydrostatic cell has been constructed but not yet used.

The second part of this thesis deals with a study of the Co²⁺ ® Co³⁺ insulin transition upon oxidation in H₂O₂, using semi-time resolved cryocrystallography. As well as the two end states, three possible intermediate states were examined. Difference electron density maps between the soaked states and Co²⁺ insulin indicated a conversion to the unstable tetrahedral Co³⁺ form in the intermediate states, with associated protein conformation changes, before reverting back to the stable octahedral Co²⁺ state.

The results from the high pressure studies indicate that genuine unit cell changes occur at elevated hydrostatic pressure, possibly indicative of a transition or intermediate state. Semi-time resolved cryocrystallography has proved a useful tool for investigations into slow protein transitions and their associated intermediate states.