Characterizing Neurosteroid Binding in the GABA(A) Receptor Using Top-Down Mass Spectrometry

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Dr. Wayland Cheng is a cardiothoracic anesthesiologist with the long-term goal of being an independent investigator in anesthetic pharmacology. His research background is in the field of ion channel biophysics, and his interest is in the interaction of anesthetics with pentameric ligand gated ion channels (pLGICs) with a focus on neurosteroids. Neurosteroids are endogenous brain sterols that potently modulate GABAA receptors (GABAARs), and are being developed as anesthetics or treatments of neurologic and psychiatric disorders. Although neurosteroid modulation of the GABAAR has been well characterized, neurosteroid binding to the GABAAR is not well understood. A detailed understanding of the stoichiometry/site(s), orientation and pharmacology of neurosteroid binding to the GABAAR is essential for structure-based drug design. To address this deficiency, the candidate will apply photo-affinity labeling with an existing library of neurosteroid photolabeling reagents, and top-down mass spectrometry (MS), a technique that analyzes intact membrane proteins, to characterize neurosteroid photolabeling of the GABAAR. Aim 1 is to determine the stoichiometry/site(s), orientation and pharmacophore of neurosteroid photolabeling of ELIC-GABAAR chimeras (tractable model proteins where the transmembrane domain consists of GABAAR subunits). Aim 2 is to determine the stoichiometry of neurosteroid photolabeling of α1β3γ2 GABAARs proteoforms (intact proteins and all associated modifications), and to test whether certain proteoforms are preferentially photolabeled by neurosteroids. This work will yield novel insights into the structural basis of neurosteroid binding to the GABAAR. It will also establish a generalizable method using top-down MS for characterizing small molecule photolabeling of membrane proteins, and is anticipated to lead to sustainable R01-level funding. This research will be conducted under the mentorship of Dr. Alex Evers, an expert in anesthetic pharmacology, photo-affinity labeling and bottom-up mass spectrometry. The candidate will also be working closely with Dr. Michael Gross and Dr. Julian Whitelegge, both renowned leaders in top-down MS of membrane proteins. The Anesthesiology Department at Washington University provides a robust environment for research in molecular pharmacology. Furthermore, the candidate utilizes an Elite mass spectrometer owned by Dr. Evers, and has full access to the Washington University NIH NIGMS Mass Spectrometry Research Resource led by Dr. Gross, which provides state-of-the-art MS technology for high mass and high resolution measurements.