CHANGES IN CONFORMATION OF PHOSPHORYLATED PROTEINS

蛋白质磷酸化被认为可以控制广泛的细胞事件，如新陈代谢、细胞分裂、膜运输和基因表达等。然而，磷酸化调控的生物物理基础只在两个系统中被研究过。结合光谱-分子模拟的方法将提供必要的结构信息，以帮助理解磷酸化在蛋白质功能中的抑制或激活效应。抑制或激活蛋白质功能的一个可能机制是，磷酸化可能导致构象的变化，从而影响其与其他蛋白质或生物分子的相互作用。这项研究中使用的一种模型蛋白质是中心素。Centrin是一种普遍存在的蛋白质，与中心粒/基体、中心体和有丝分裂纺锤体极有关，因此是负责将染色体分配给子细胞的微管组织中心(MTOC)的组成部分。最近的研究结果表明，在细胞分裂过程中，中心体复制对其磷酸化形式的中心蛋白具有激活作用。我们的目标是：(1)在细菌系统中过量表达人类1，2和衣藻中心蛋白，从而为生物物理研究提供丰富的蛋白质来源。(2)采用不同的层析技术分离和鉴定Centin，并通过SDS-PAGE、蛋白质印迹分析、氨基酸序列分析和飞行时间(TOF)质谱仪确定其纯度。(3)在体外利用蛋白激酶A(PKA)和三磷酸腺苷(ATP)对Centin进行足够数量的磷酸化，以进行生物物理表征。(4)用傅立叶变换红外光谱(FT-IR)和圆二色谱(CD)研究了磷酸化和非磷酸化的中心素构象。(5)用二维红外相关法比较蛋白质磷酸化和非磷酸化状态的光谱。(6)通过分子模拟技术对这些结果进行模拟。这种研究磷酸化中心素的跨学科方法将使我们能够建立受磷酸化调控的蛋白质的构象和功能之间的关系。这项研究的意义将是提供分子水平的信息，这些信息将有助于理解所研究的细胞事件，如细胞分裂和细胞分裂失控，如细胞癌。长期目标是利用振动光谱学研究定点突变产生的变异蛋白质，并通过核磁共振确定这些蛋白质的结构。

Protein phosphorylation is known to control a wide range of cellular events such as: metabolism, cell division, membrane transport and gene expression among others. However, the Biophysical basis of regulation by phosphorylation has only been studied in two systems. A combined spectroscopic-molecular modeling approach will provide the necessary structural information to aid in the understanding of the inhibitory or activating effect of phosphorylation in protein function. One possible mechanism for the inhibitory or activating effects on protein function is that phosphorylation may induce a change in conformation and thus effect its interaction with other proteins or biological molecules. A model protein used in this study is centrin. Centrin has been found to be a ubiquitous protein associated with centrioles/basal bodies, centrosomes, and mitotic spindle poles and therefore is an integral component of the microtubule organizing center (MTOC) which is responsible for distributing the chromosomes to the daughter cells. Recent findings suggests an activating effect on centrin in its phosphorylated forms which is essential in centrosomal duplication during cell division. Our aims are: (1) To over-express Human 1, 2 and Chlamydomonas centrin in a bacterial system allowing for an abundant source of protein required for the biophysical study. (2) To isolate and characterize centrin using various chromatographic techniques and conform its purity via SDS-PAGE, western blot analysis, amino acid sequence and time of flight (TOF) mass spectrometry. (3) To phosphorylate centrin in vitro using protein kinase A (PKA) and adenosine triphosphate (ATP) in sufficient quantities for Biophysical characterization. (4) To investigate phosphorylated and un-phosphorylated centrin conformation using Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD). (5) To use two-dimensional infrared correlation for the comparison of spectra obtained for proteins in their phosphorylation and un-phosphorylated states. (6) To model these results via molecular modeling techniques. This inter-disciplinary approach to the study of phosphorylated centrin will enable us to establish a relationship between conformation and function in a protein regulated by phosphorylation. The implications of such a study will be to contribute molecular level information which will aid in the understanding of the cellular events studied such as cell division and uncontrolled cell division such as in cell carcinomas. Long-term goals are to study variant proteins generated by site-specific mutagenesis using vibrational spectroscopy and to determine the structure of these proteins via NMR.