Structural analysis of prion proteins and mechanism of PrPsc transition by using a novel dynamic molecular structure analysis

我们利用SDSL-ESR技术检测了D177N（人类为178N）突变对不同ph值的moPrPc S2区构象稳定性的影响。我们制备了与甲烷硫代磺酸自旋探针（Y161R1和Y161R1/D177N）反应的moPrPc突变体。D177N在pH 7.5时的ESR谱较Y161R1窄，记为WT^*。当溶液pH从7.5降低到4.0时，D177N的ESR谱没有变化。这些结果表明，盐桥（D177-R163）的消失导致S2区的不稳定性增加。从WT^*得到的ESR光谱中，中心组分（Mi=0）的1/H值在pH为7.5 ~ 6.5时保持不变，而当溶液pH降至6.0以下时，1/Ho值突然增加。这些结果表明，在pH 6.5 ~ 6.0之间存在从刚性结构到柔性结构的构象转变。此外，在pH 7.5时，与S2区连接的盐桥相邻的H176S获得的ESR谱线形比WT^*窄。当溶液pH从7.5降低到4.0时，H176S的光谱变化很小。这些结果表明，h176的质子化与S2区的稳定性密切相关。这些发现对于理解S2区盐桥的破坏在遗传性克雅氏病和致命性家族性失眠症中形成致病性PrP_Sc结构的机制具有重要意义。

We examined the influence of D177N (178N in humans) mutation on the conformational stability of the S2 region of moPrPc with varying pHs by using the SDSL-ESR technique. We prepared moPrPc mutants that reacted with methane thiosulfonate spin-probes (Y161R1 and Y161R1/D177N). The ESR spectrum of D177N at pH 7.5 was narrower than that of Y161R1, referred to as WT^*. The ESR spectrum of D177N did not change when pH in the solution decreased from 7.5 to 4.0.These results suggested that the disappearance of a salt bridge (D177-R163) induced the increase in the instability of S2 region. The values of 1/ H of the central component (Mi=0) in the ESR spectrum obtained from WT^* remained constant from pH 7.5 to pH 6.5, whereas an abrupt increase of 1/Ho occurred when the pH in the solution decreased to under 6.0. These findings indicated that the conformational transition from a rigid structure to a flexible structure existed at between pH 6.5 and pH 6.0. Moreover, the line shape of the ESR spectrum obtained from H176S neighboring the salt bridge linked to the S2 region was narrower than that of WT^* at pH 7.5. When the pH in the solution decreased from 7.5 to 4.0, the change in the spectrum of H176S was small. These results indicate that the protonation of H 176 is strongly associated with the stability of S2 region. These findings are important for understanding the mechanism by which the disruption of the salt bridge in the S2 region forms the pathogenic PrP_Sc structure in hereditary Creutzfeldt-Jacob disease and fatal familial insomnia.