Project Summary The Evers laboratory studies the binding interactions of neurosteroids and cholesterol with integral membrane proteins, with the aim of identifying the specific binding events underlying sterol modulation of protein function. Our major focus is on neurosteroid (NS) modulation of -aminobutyric acid type A receptors (GABAA- R). Neurosteroids are important modulators of neuronal excitability and nervous system development with enormous therapeutic potential as anesthetics, anti-depressants and neuro-protectants. We have shown that there are multiple, subunit-specific binding sites for neurosteroids on GABAA receptors, each of which contributes to the functional effects of neurosteroids. In the proposed research, we will use photolabeling techniques to define the precise sites at which the major classes of neurosteroids bind on the most abundant forms of synaptic and extra-synaptic GABAA receptors and determine the functional significance of each identified binding site by assessing the effect of targeted amino acid substitutions on NS modulation of GABAA- R currents. To identify photo-labeled residues we will utilize state-of-the-art protein chemistry and expression techniques in conjunction with cutting edge mass spectrometry (MS) methods, including middle-down and intact protein MS. High-resolution cryogenic-electron microscopy structures will be obtained to identify the atomic details of novel NS binding sites and to investigate binding interactions that appear to stabilize conformations not captured in current structures. Fluorescence-based binding assays will then be used to measure the site-specific affinity of various NS for the identified binding sites. These assays will be adapted to stopped-flow fluorimetry to determine the state-dependence of binding and to a plate reader format to screen for site-specific agonists and antagonists. The long term goal of our NS program is to develop and use site-specific NS ligands to probe the role of specific NS binding sites and GABAA-R subtypes in the behavioral effects of endogenous NS and the mechanisms of action of NS sedatives and anesthetics. We have also used cholesterol-analogue photolabeling to identify specific binding sites that mediate cholesterol inhibition of the lipid scramblase, nhTMEM16, and cholesterol modulation of mTOR1 by the lysosomal membrane protein GPR155. Both nhTMEM16 and GPR155 have two specific cholesterol binding sites per protein monomer and we are using targeted amino acid substitution to understand the functional role of each site. We are also developing fluorescence-based binding assays to measure cholesterol affinity and sterol specificity for these sites. Novel cholesterol binding sites present new targets for small molecule allosteric modulators of membrane protein function and the tools we have developed are widely applicable to identifying binding sites on other cholesterol-modulated proteins and screening for site-specific ligands.