The Small-Angle X-Ray Scattering from Proteins Under Pressure
The overall solution structures of proteins under pressure were studied by using the synchrotron small-angle X-ray scattering (SAXS) technique. The measurements were made with a hydrostatic pressure cell with diamond windows, which enabled quantitative analysis of scattering profiles. Two applications were demonstrated: the pressure-induced denaturation of a monomeric protein, myoglobin, and the dissociation of an oligomeric protein, lactate dehydrogenase (LDH). Myoglobin showed sigmoid transition with a mid-point at 180 MPa. Denatured myoglobin at 300 MPa gave 20.9±0.9Å for the radius of gyration (Rg), which enabled quantitative comparison with different unfolding states. Pressure-denatured myoglobin at acidic pH was much more compact than myoglobin in urea-induced unfolded state and even more compact than myoglobin in a molten globule state. LDH consists of four identical subunits. Forward scattering, I(0)/C, which is proportional to molecular weight, showed that LDH dissociated into not monomers but dimer subunits with applied pressure. The conformational drift was confirmed by the value of Rg. There is little structural difference between native and drifted LDH. The presence of five scattering peaks in the medium-angle region indicates the dissociated dimer does not take a molten-globule-like structure but instead a structure in which the core is retained. The analysis of SAXS profiles under high pressure enabled modeling of the orientation of dimer subunits, which seems to be chosen so as to reduce the volume without disrupting the core structure.
KeywordsDenature State Oligomeric Protein SAXS Measurement Staphylococcal Nuclease Guinier Plot
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