PHOSPHORYLATION EFFECTS ON THE FOLDING OF CHLAMYDOMONAS CENTRIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 Centrin是一种钙结合蛋白，定位于脊椎动物细胞的中心粒中。中心素的磷酸化已被证明是中心体前期分离的信号，并与中心粒分离有关。Sanoguet及其同事最近的工作研究了中心素磷酸化对蛋白质热稳定性和结构动力学的影响。利用傅里叶变换红外光谱和二维相关光谱，他们能够确定由于热变性引起的磷酸化和全息中心蛋白的结构变化。结果表明，磷酸化影响C-末端结构域的环的展开。此外，有证据表明存在涉及酰胺I天冬氨酸带的预跃迁。鉴于这些发现，我们建议使用激光诱导温度跳跃与红外光谱相结合的方法来监测磷酸化中心蛋白和全中心蛋白C-末端结构域的折叠动力学。Centin的C-末端结构域在结构上独立于其他结构域，仅对该片段的研究将简化实验及其解释。实验的目的是监测天冬氨酸的振动模式，它显示了低温下前跃迁吸收的变化，以及环状结构的振动，它显示了高温变性。对这些结构变化的磷酸化和全集中素动力学进行比较，可能会更好地理解中心素的生物学功能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Centrin is a calcium binding protein which is localized in centrioles in vertebrate cells. The phosphorylation of centrin has been shown to signal the separation of centrosomes at prophase and has been implicated in centriole separation. Recent work by Sanoguet and coworkers has investigated the effect of centrin phosphorylation upon the protein thermal stability and structural dynamics. Using Fourier transform infrared spectroscopy and two-dimensional correlation spectroscopy, they were able to determine the structural changes due to thermal denaturation for both phosphorylated and holo-centrin. The results indicated that phosphorylation affects the unfolding of the loops of the C-terminal domain. Furthermore, there is evidence of a pre-transition involving the aspartate band of the Amide I. In light of these findings, we propose to use the method of Laser Induced Temperature-Jump in conjunction with infrared spectroscopy to monitor the folding dynamics of the C-terminal domain of phosphorylated centrin and holo-centrin. The C-terminal domain of centrin is structurally independent from other domains, and study of this fragment alone will simplify the experiment and its interpretation. The experiments will aim to monitor the aspartate vibrational mode which shows pre-transition absorption changes at low temperatures as well as the vibrations of the loop structures which show high temperature denaturation. A comparison of the phosphorylated and holo-centrin kinetics for these structural changes may yield a greater understanding of the biological function of centrin.