Single-molecule Measurements of Membrane-protein Folding and Ligand-Interaction Energetics in Bacteriorhodopsin and the Diabetes-insipidus-involved Vasopressin Receptor 2

细菌视紫红质和尿崩症相关加压素受体 2 中膜蛋白折叠和配体相互作用能量的单分子测量

• 批准号: 10356067
• 负责人: David R Jacobson
• 金额: $ 8.66万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356067
• 关键词: Atomic Force Microscopy; Bacteriorhodopsins; Binding; Biochemical; Biological Assay; Biological Models; Cell surface; Chemicals; Colorado; Detergents; Diabetes Insipidus; Disease; Drug Antagonism; Environment; Free Energy; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; Geometry; Goals; Human; Ligand Binding; Ligands; Light; Link; Lipid Bilayers; Lipids; Measurement; Measures; Membrane Proteins; Mentors; Methods; Micelles; Molecular; Molecular Chaperones; Molecular Conformation; Movement; Mutation; N-terminal; Nucleic Acids; Pharmaceutical Preparations; Pharmacology; Phase; Photons; Point Mutation; Proline; Protein Biochemistry; Protein Dynamics; Proteins; Residual state; Retina; Sampling; Scanning Probe Microscopes; Site; Structure; System; Techniques; Testing; Thermodynamics; Time; Training; Universities; Urine; Use of New Techniques; Vasopressin Antagonist; Vasopressin Receptor; Vasopressins; Work; X ray diffraction analysis; absorption; alpha helix; argipressin receptor; base; collecting tubule structure; comparative; disease-causing mutation; experimental study; insight; membrane model; mutant; non-Native; protein folding; response; single molecule; technique development; vasopressin resistant diabetes insipidus

Project Summary/Abstract Human arginine vasopressin receptor 2 (AVPR2) is an -helical membrane protein expressed in the collecting ducts of the kidneys involved in regulating urine volume. Mutations of AVPR2 at some 96 sites are known to cause nephrogenic diabetes insipidus (NDI), likely by promoting misfolding. The vasopressin antagonist drugs (“vaptans”) have been shown to rescue cell-surface expression, putatively by acting as chaperones stabilizing the native folded state. Thus, understanding the relative energetics of the folded and misfolded states in the presence and absence of ligands would shed light on the native structural dynamics of AVPR2 and how those dynamics are changed by disease-causing mutations. Such results would be relevant to NDI, and also more generally to diseases arising from G-protein coupled receptors (GPCRs). However, the established biochemical technique of chemical denaturation in detergent micelles that is used to measure membrane-protein thermodynamic stability (G) and its change upon mutation or ligand binding (G) suffers from several limitations that make it unsuitable for studies of AVPR2. In particular, the non-native detergent environment, the poorly defined denatured state with significant residual secondary structure, and the need to extrapolate from high denaturant concentration cause the measured energetics to poorly reflect the underlying, biologically relevant molecular values. Most significantly, chemical-denaturation-based techniques have never been successfully applied to GPCRs because GPCRs do not globally refold when the denaturant is removed. These shortcomings motivate the overall aim of this proposal: to develop alternate techniques for measuring membrane-protein energetics, based on force-induced unfolding rather than chemical denaturation. Such techniques, implemented on an atomic force microscope (AFM), can study membrane proteins in the native lipid bilayer and obviate the problem of globally reversible unfolding by probing a small portion of the protein at a time. Work during the postdoctoral K99 phase will use the model membrane protein bacteriorhodopsin (bR) to further develop these force-based techniques. Two particular aims will be achieved: (1) measurement of point-mutant free energy changes of bR in its native bilayer and without confounding chemical denaturant and (2) quantification of the energetics of a photo-activated ligand isomerization in bR. Completing this work during the K99 phase will establish the basis for the aim of the independent R00 phase: to elucidate the folding and ligand-interaction energetics of AVPR2 using these new techniques. In addition to providing specific insight into AVPR2 folding and misfolding, this work will establish a new paradigm in which energetic measurements can be made directly in biomedically relevant systems like AVPR2, rather than just in model systems. The transition to independence will also be facilitated by training during the K99 phase, most notably in the expression and purification of GPCR samples. The University of Colorado provides world-class facilities for carrying out this work, and co-mentors will offer expertise in both single-molecule AFM experiments and membrane-protein biochemistry.

项目概要/摘要 人精氨酸加压素受体 2 (AVPR2) 是一种 α 螺旋膜蛋白，表达于收集细胞中。 肾脏的管道参与调节尿量。已知 AVPR2 在约 96 个位点发生突变 可能通过促进错误折叠引起肾性尿崩症（NDI）。加压素拮抗剂药物 （“vaptans”）已被证明可以通过充当稳定细胞表面的伴侣来拯救细胞表面表达 原生折叠状态。因此，了解存在的折叠和错误折叠状态的相对能量学 配体的缺失将揭示 AVPR2 的天然结构动力学以及这些动力学如何 因致病突变而改变。这样的结果将与 NDI 相关，并且更普遍地与 由 G 蛋白偶联受体 (GPCR) 引起的疾病。然而，现有的生化技术 用于测量膜蛋白热力学稳定性的洗涤剂胶束中的化学变性 (G) 及其在突变或配体结合时的变化 (G) 受到一些限制，使其 不适合 AVPR2 的研究。特别是，非天然洗涤剂环境，定义不明确 具有显着残余二级结构的变性状态，并且需要从高变性剂推断 浓度导致测量的能量不能很好地反映潜在的、生物学相关的分子 价值观。最重要的是，基于化学变性的技术从未成功应用于 GPCR，因为当变性剂被去除时，GPCR 不会全局重折叠。这些缺点促使 该提案的总体目标：开发测量膜蛋白能量学的替代技术，基于 力诱导的展开而不是化学变性。这些技术，在原子上实现 力显微镜（AFM），可以研究天然脂质双层中的膜蛋白并消除问题 通过一次探测一小部分蛋白质来实现全局可逆的展开。博士后期间工作 K99阶段将使用模型膜蛋白细菌视紫红质（bR）来进一步开发这些基于力的 技术。将实现两个特定目标：（1）测量 bR 的点突变自由能变化 其天然双层且不混杂化学变性剂和（2）能量学的量化 bR 中的光激活配体异构化。在K99阶段完成这项工作将为奠定基础 独立 R00 相的目的：阐明 AVPR2 的折叠和配体相互作用能量学 使用这些新技术。除了提供对 AVPR2 折叠和错误折叠的具体见解之外，这 这项工作将建立一个新的范式，在该范式中可以直接在生物医学中进行能量测量 AVPR2 等相关系统，而不仅仅是模型系统。向独立的过渡也将是 K99 阶段的培训促进了这一过程，最显着的是 GPCR 样品的表达和纯化。 科罗拉多大学为开展这项工作提供世界一流的设施，共同导师将提供 单分子 AFM 实验和膜蛋白生物化学方面的专业知识。