Structural studies of conversion of prion protein and crystallographic studies of superoxide dismutase from Pyrobaculum aerophilum.

摘要

Conversion of prion protein from the normal, cellular form, PrP C, to the pathological, infectious form, PrPSc, is the key event in the pathogenesis of transmissible spongiform encephalopathies, fatal neurodegenerative diseases. To understand the molecular basis of the conversion of prion protein, an in vitro conversion system using recombinant hamster prion protein was developed and characterized.; Crystal twinning is detrimental to structure determination by X-ray diffraction. Practical strategies to detect and overcome crystal twinning are presented with the structure of superoxide dismutase from Pyrobaculum aerophilum .; To obtain pure and stable recombinant hamster prion protein for structural studies, several constructs covering regions of hamster prion protein were studied. Constructs encoding hamster prion protein with a hexa-histidine tag are characterized by biophysical and biochemical methods. These constructs were found to be monomeric in solution and have characteristics of PrP C.; We developed in vitro conversion system where recombinant hamster prion protein was converted to a second form, PrPRDX, by a reduction-oxidation process. PrpRDX seeds conversion of PrPC to PrPRDX. PrpRDX shares properties with PrPSc such as oligomerization, seeded conversion, protease resistance and enhance β-sheet content. A domain-swapping model involving intermolecular disulfide bonds is proposed to account for experimental observations and general properties of prions.; A domain-swapping model for PrpRDX fibril with two variations is proposed in details. The PrpRDX model features two β-sheets separated by 11 Å at the center of PrpRDX fibril surrounded by globular domains that are swapped by intermolecular disulfide bonds. The two sheet feature of the PrpRDX model was validated by matching X-ray diffraction data with simulated data based on slit models.; As a part of a pilot structural genomics project, the structure of superoxide dismutase from Pyrobaculum aerophilum was determined at 1.8 Å resolution by molecular replacement. The structure presents challenges in structure determination because of multiple molecules in the asymmetric unit, pseudo-symmetry and crystal twinning. Practical strategies to detect and deal with crystal twinning in the presence of non-crystallographic symmetry are presented. The structure represents the metal-free form.