Keiichi Kameyama

Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

The small heat shock proteins (sHSPs) solubilize thermo-denatured proteins without adenosine triphosphate energy consumption to facilitate protein refolding. sHSP20.8 is one of the silkworm (Bombyx mori) sHSPs having only one cystein in the N-terminal domain: Cys43. We report a simple measurement of oligomeric transition of sHSP20.8 using high hydrostatic pressure native polyacrylamide gel electrophoresis (high hydrostatic pressure (HP) native polyacrylamide gel electrophoresis (PAGE)). At ambient pressure under oxydative condition, the native PAGE of thermal transition of sHSP20.8 oligomer displayed a cooperative association. In contrast, HP native PAGE clearly demonstrated that sHSP20.8 dissociated at 80 MPa and 25°C, and the resultant molecular species gradually reassociated with time under that condition. In addition, the reassociation process was suppressed in the presence of the reductant. These results are consistent with the idea that sHSP20.8 oligomer temporally dissociates at the first thermo-sensing step and reassociates with the oxidation of Cys43.

Negative thermal expansibility change for dissociation of lysozyme variant amyloid protofibril

A disulfide‐deficient variant of hen lysozyme, 0SS, is known to form an amyloid protofibril spontaneously, and to dissociate into monomers at high hydrostatic pressure. We carried out native PAGE at various temperatures (20–35°C) and pressures (0.1–200 MPa), to characterize the dissociation equilibrium of disulfide‐deficient variant of hen lysozyme amyloid protofibril. Based on the density profiles, the partial molar volume and thermal expansibility changes for dissociation, ΔvD and ΔeD, were obtained to be −74 cm3/mol at 25°C and −2.3 cm3 mol−1 K−1, respectively. The dissociation of amyloid fibril destroys the cross β‐structure, and such conformational destruction in native protein fold rarely accompanies negative thermal expansibility change. We discussed the negative thermal expansibility change in terms of hydration and structural packing of the amyloid protofibril.