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

有了这个奖项，生命过程的化学计划正在资助斯托尼布鲁克大学化学系的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。 该项目由BIO董事会分子和细胞生物科学部的生物分子动力学，结构和功能集群共同资助。靶向蛋白质是水溶性的界面酶或调节性大分子，其在催化反应或信号事件的过程中瞬时结合到膜表面。表征活性蛋白质复合物在膜界面的构象是结构生物学中的主要挑战。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发生故障非常重要。 初步数据表明，用季铵部分修饰的马来酰亚胺稳健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开发，校准，并建立其效用。这些研究将提供有关蛋白质残基参与膜结合的性质的信息。这种方法将开发与胆固醇氧化酶链霉菌，因为它很容易纯化和表达，并服从质谱和光谱表征。在更广泛的影响方面，希望这种生物有机/质谱方法将是便携式的，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。使用廉价的，易于合成的半胱氨酸标记试剂，使蛋白质膜结合的分析广泛可用的MALDI-TOF质谱仪应促进这一点。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经验。桑普森教授将继续她在当地小学的活动，其中包括组织化学演示主要由研究生在每年的K-5科学博览会，其中超过150名儿童每年参加。

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.