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

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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.