CHANGES IN CONFORMATION OF PHOSPHORYLATED PROTEINS

磷酸化蛋白质构象的变化

• 批准号: 6107151
• 负责人: Belinda Pastrana-Rios
• 金额: $ 5.66万
• 依托单位: UNIVERSITY OF PUERTO RICO MAYAGUEZ
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107151
• 关键词: Chlamydomonas; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; biophysics; cell cycle; chemical models; circular dichroism; conformation; human tissue; infrared spectrometry; mass spectrometry; nuclear magnetic resonance spectroscopy; phosphoproteins; phosphorylation; plant proteins; protein kinase A; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; ubiquitin; western blottings

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.

已知蛋白质磷酸化可以控制广泛的细胞事件，例如：代谢，细胞分裂，膜转运和基因表达等。但是，仅在两个系统中研究了通过磷酸化调节的生物物理基础。综合光谱分子建模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作用或激活作用。抑制性或激活对蛋白质功能的影响的一种可能机制是，磷酸化可能诱导构象的变化，从而影响其与其他蛋白质或生物分子的相互作用。本研究中使用的模型蛋白是中心蛋白。已发现中心蛋白是一种与中心/基体，中心体和有丝分裂纺锤体有关的无处不在蛋白质，因此是微管组织中心（MTOC）的组成部分，该组成部分负责将染色体分配给子细胞。最近的发现表明，其磷酸化形式的中心素有激活作用，这对于细胞分裂期间的中心体重复至关重要。我们的目的是：（1）在细菌系统中对过表达的人1、2和衣原体中心素，从而允许生物物理研究所需的大量蛋白质来源。 （2）使用各种色谱技术隔离和表征中心蛋白，并通过SDS-PAGE，Western印迹分析，氨基酸序列和飞行时间（TOF）质谱法符合其纯度。 （3）使用蛋白激酶A（PKA）和三磷酸腺苷（ATP）在体外磷酸化中心蛋白，以足够的量进行生物物理表征。 （4）研究使用傅立叶变换红外（FT-IR）光谱和圆形二分（CD）研究磷酸化和未磷酸化的中心蛋白构象。 （5）使用二维红外相关性来比较其在其磷酸化和非磷酸化态中蛋白质获得的光谱。 （6）通过分子建模技术对这些结果进行建模。磷酸化中心蛋白研究的这种跨学科方法将使我们能够在受磷酸化调节的蛋白质中建立构象与功能之间的关系。这项研究的含义将是贡献分子水平信息，这将有助于理解所研究的细胞事件，例如细胞分裂和不受控制的细胞分裂，例如细胞癌。长期目标是研究使用振动光谱学特定诱变产生的变异蛋白，并通过NMR确定这些蛋白质的结构。