Synthesis and Evaluation of ICMT Reagents for Mapping Lipid-Protein Interactions

用于绘制脂质-蛋白质相互作用的 ICMT 试剂的合成和评价

• 批准号: 1058349
• 负责人: Nicole Sampson
• 金额: $ 40万
• 依托单位: SUNY at Stony Brook
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1058349&HistoricalAwards=false
• 关键词: Synthesis; Evaluation; ICMT; Reagents; Mapping

With this award, the Chemistry of Life Processes Program is funding Professor Nicole Sampson of the Department of Chemistry at Stony Brook University to develop an isotope coded mass tagging approach to study interfacial proteins. This project is being co-funded by the Biomolecular Dynamics, Structure and Function Cluster in the Molecular and Cellular Biosciences Division of the BIO Directorate. The targeted proteins are water-soluble, interfacial enzymes or regulatory macromolecules that bind transiently to the membrane surface during the course of the catalytic reaction or signaling event. Characterizing the conformation of the active protein complex at the membrane interface is a major challenge in structural biology. These structures are important for understanding how these proteins work under physiological conditions and how they malfunction in disease. Preliminary data demonstrate that maleimide modified with a quaternary ammonium moiety robustly labels a protein containing multiple cysteine residues. In this research project, maleimide probes will be further developed, calibrated, and their utility established. These studies will provide information about the nature of protein residues involved in membrane binding. This methodology will be developed with cholesterol oxidase from Streptomyces because it is easily purified and expressed, and is amenable to mass and spectroscopic characterization. In terms of Broader Impacts, it is hoped that this bioorganic/mass spectrometric method will be portable, and provide guidance to members of the community who wish to perform complementary structural analyses of other membrane-bound proteins to determine active protein structures at the membrane interface. The use of inexpensive, readily-synthesized cysteine labeling reagents that enable analysis of protein-membrane binding on widely-available MALDI-TOF mass spectrometers should facilitate this. The project will offer an interdisciplinary research experience to undergraduate, graduate, and postdoctoral fellow coworkers. Professor Sampson will continue her activities in the local elementary school that include organizing chemistry demonstrations performed primarily by graduate students at the annual K-5 science fair in which more than 150 children participate every year.

有了这个奖项，生命过程化学项目将资助石溪大学化学系的Nicole Sampson教授开发一种同位素编码的质量标记方法来研究界面蛋白质。该项目由生物技术理事会分子和细胞生物科学部的生物分子动力学、结构和功能组共同资助。目标蛋白是在催化反应或信号事件过程中短暂结合膜表面的水溶性、界面酶或调节性大分子。表征活性蛋白复合物在膜界面上的构象是结构生物学的一个主要挑战。这些结构对于理解这些蛋白质在生理条件下如何工作以及它们在疾病中如何失灵非常重要。初步数据表明，用季铵盐修饰的马来酰亚胺可以有效地标记含有多个半胱氨酸残基的蛋白质。在这个研究项目中，马来酰亚胺探针将进一步发展，校准，并建立其实用性。这些研究将提供有关参与膜结合的蛋白质残基性质的信息。该方法将利用链霉菌中的胆固醇氧化酶进行开发，因为它易于纯化和表达，并且易于进行质谱和光谱表征。就更广泛的影响而言，希望这种生物有机/质谱方法具有可移植性，并为希望对其他膜结合蛋白进行互补结构分析以确定膜界面活性蛋白结构的社区成员提供指导。使用廉价、易于合成的半胱氨酸标记试剂，可以在广泛使用的MALDI-TOF质谱仪上分析蛋白质-膜结合，这将有助于实现这一目标。该项目将为本科生、研究生和博士后同事提供跨学科的研究经验。Sampson教授将继续她在当地小学的活动，包括在每年有150多名儿童参加的K-5科学博览会上组织主要由研究生进行的化学演示。