PHOSPHORYLATION EFFECTS ON THE FOLDING OF CHLAMYDOMONAS CENTRIN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。中心蛋白是脊椎动物细胞中心粒中的钙结合蛋白。中心蛋白的磷酸化已被证明是中心体在前期分离的信号，并已被牵连在中心粒分离。Sanoguet及其同事最近的工作研究了中心蛋白磷酸化对蛋白质热稳定性和结构动力学的影响。使用傅里叶变换红外光谱和二维相关光谱，他们能够确定由于磷酸化和holo-centrin的热变性引起的结构变化。结果表明，磷酸化影响C-末端结构域环的展开。此外，存在涉及酰胺I的天冬氨酸谱带的预跃迁的证据。鉴于这些发现，我们建议使用激光诱导温度跳跃结合红外光谱的方法来监测磷酸化中心蛋白和holo-centrin的C-末端结构域的折叠动力学。中心蛋白的C-末端结构域在结构上独立于其他结构域，单独研究该片段将简化实验及其解释。实验的目的是监测天冬氨酸振动模式，该模式在低温下显示过渡前吸收变化，以及环结构的振动，该振动显示高温变性。这些结构变化的磷酸化和holo-centrin动力学的比较，可能会产生一个更好的了解centrin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.