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

通过这一奖项，生命过程化学计划资助石溪大学化学系的Nicole Sampson教授开发一种同位素编码质量标记方法来研究界面蛋白质。该项目由生物局分子和细胞生物科学部的生物分子动力学、结构和功能集群共同资助。靶蛋白是在催化反应或信号传递过程中瞬间结合到膜表面的水溶性、界面酶或调节性大分子。表征膜界面活性蛋白复合体的构象是结构生物学中的一个主要挑战。这些结构对于了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，用季铵盐基修饰的马来酰亚胺牢固地标记了含有多个半胱氨酸残基的蛋白质。在这项研究项目中，马来酰亚胺探头将进一步开发、校准，并建立其实用价值。这些研究将提供与膜结合有关的蛋白质残基的性质的信息。这种方法将与来自链霉菌的胆固醇氧化酶一起开发，因为它易于纯化和表达，并且易于进行质量和光谱表征。就更广泛的影响而言，希望这种生物有机/质谱学方法能够便携，并为希望对其他膜结合蛋白质进行互补结构分析以确定膜界面活性蛋白质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，可以在广泛可用的MALDI-TOF质谱仪上分析蛋白质膜结合，应该会促进这一点。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体验。桑普森教授将继续她在当地小学的活动，包括组织主要由研究生在一年一度的K-5科学博览会上表演的化学演示，每年有150多名儿童参加。

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.