| The majority of the biosphere is above 100 atmospheres. Pressures present at ocean floors, over a thousand atmospheres, affect kinetic and equilibrium constants, multimeric association and ligand binding, membrane permeability and ion transduction, cellular metabolism, viral infectivity, and protein folding. Pressure gives information on volume fluctuations, important in protein function. Pressure also has biotechnological applications, for example, in sterilizing food products and in modulating sensitivity of biomedical assays.; Intrinsic compressibilities of globular proteins are small (less than that of water). Thus knowledge of the arrangement of atoms within proteins is critical in understanding large observed effects. Unfortunately, the probe most powerful at giving atomic-level information is the one least developed for high pressures - x-ray crystallography. To date, only one structure is solved under thousand atmosphere-range pressures [Kundrot, C. E. & Richards, F. M. (1987) Journal of Molecular Biology. 193, 157--170]. The main difficulty is the presence of strong diffraction rings at crystallographic ally important resolutions, due to the use of a beryllium pressure cell.; To surmount difficulties from beryllium, we attempt to lock-in pressure induced changes by cooling protein crystals under pressure to below the glass transition temperature. Diffraction is performed at low temperatures and ambient pressure.; Structural changes in sperm whale myoglobin upon pressurization at room temperature and changes observed upon cooling under high pressure show good correlation, indicating major structural rearrangements have indeed been frozen-in. Changes involve an anisotropic rearrangement of secondary structures, such as alpha helices.; Next, because protein crystals have a high solvent content, ice formation during cooling can cause loss of crystalline order. Yet cooling is an important technique in protecting crystals from radiation damage during diffraction. The high pressure cooling method presented here may be an alternative in cases where de facto flash cooling is inadequate.; Finally, observed structural changes in myoglobin, when combined with high pressure optical studies performed previously by other researchers, provide evidence for a ligand pathway to the active site through the protein matrix, hence demonstrating the utility of high pressure crystallographic techniques. |
