Neurosteroid and Cholesterol Binding to Integral Membrane Proteins

项目摘要 埃弗斯实验室研究神经甾体和胆固醇与完整膜的结合相互作用 蛋白质，目的是确定蛋白质功能的固醇调节背后的特异性结合事件。 我们的主要重点是神经甾体（NS）的α-氨基丁酸A型受体（GABAA-）的调制。 R）。神经类固醇是神经元兴奋性和神经系统发育的重要调节剂， 作为麻醉剂、抗抑郁剂和神经保护剂具有巨大的治疗潜力。我们已经表明 GABAA受体上有多个神经类固醇的亚单位特异性结合位点，每个位点都 有助于神经类固醇的功能作用。在拟议的研究中，我们将使用光标记 技术，以确定精确的网站在哪些主要类别的神经类固醇结合的最丰富的 突触和突触外GABAA受体的形式，并确定每一个的功能意义 通过评估靶向氨基酸取代对GABAA的NS调节的影响，鉴定结合位点。 R电流。为了鉴定光标记的残基，我们将利用最先进的蛋白质化学和表达 技术结合尖端质谱（MS）方法，包括中下和 将获得高分辨率低温电子显微镜结构以鉴定 新的NS结合位点的原子细节，并研究似乎稳定 在当前结构中未捕获的构象。然后，将使用基于荧光素的结合试验， 测量各种NS对所识别的结合位点的位点特异性亲和力。这些试验将 适用于停流荧光测定法，以确定结合的状态依赖性，并适用于酶标仪 格式筛选位点特异性激动剂和拮抗剂。我们NS计划的长期目标是 开发和使用位点特异性NS配体来探测特异性NS结合位点和GABAA-R亚型的作用 内源性NS的行为效应以及NS镇静剂和麻醉剂的作用机制。 我们还使用胆固醇类似物光标记来鉴定特异性结合位点， 介导脂质乱序酶nhTMEM 16的胆固醇抑制和 mTOR 1通过溶酶体膜蛋白GPR 155。nhTMEM 16和GPR 155都具有两个特异性 每个蛋白质单体的胆固醇结合位点，我们正在使用靶向氨基酸取代， 了解每个站点的功能作用。我们还在开发基于荧光的结合分析， 测量这些位点的胆固醇亲和力和固醇特异性。新的胆固醇结合位点 膜蛋白功能的小分子变构调节剂的靶点和我们拥有的工具 所开发的方法广泛适用于鉴定其他胆固醇调节蛋白上的结合位点， 筛选位点特异性配体。

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.