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.

项目总结/摘要 人精氨酸加压素受体2（AVPR 2）是一种在人外周血中表达的β-螺旋膜蛋白。 参与调节尿量的肾脏导管。已知AVPR2在大约96个位点的突变， 引起肾源性尿崩症（NDI），可能是通过促进错误折叠。血管加压素拮抗剂药物 已显示vaptans（"vaptans"）通过充当稳定细胞表面的分子伴侣来拯救细胞表面表达， 原生折叠态因此，理解存在下折叠和错误折叠状态的相对能量学， 配体的缺乏将揭示AVPR 2的天然结构动力学以及这些动力学如何 都被致病突变所改变这样的结果将与NDI相关，更普遍地说， 由G蛋白偶联受体（GPCR）引起的疾病。然而，已建立的生物化学技术， 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 突变或配体结合后的变化（突变或配体结合后的变化（突变G））受到几个限制， 不适合AVPR2的研究。特别是，非天然洗涤剂环境，定义不明确的 具有显著残留二级结构的变性状态，以及需要从高变性剂外推 浓度导致测得的能量学很难反映潜在的生物学相关分子 价值观最重要的是，基于化学变性的技术从未成功地应用于 因为当变性剂被去除时，GPCR不会整体重折叠。这些缺点促使 该提案的总体目标：开发用于测量膜蛋白能量学的替代技术， 力诱导的解折叠而不是化学变性。这种技术，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， 通过一次探测蛋白质的一小部分来进行全局可逆的解折叠。博士后工作 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.两个具体的目标将实现：（1）测量点突变的自由能变化的bR在 它的天然双层和没有混淆的化学变性剂和（2）量化的能量， bR中的光活化配体异构化。在K99阶段完成这项工作将为 对于独立的R00阶段的目的：阐明AVPR 2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了解之外， 这项工作将建立一种新的范式，可以直接在生物医学中进行能量测量 类似AVPR2的相关系统，而不仅仅是模型系统。向独立的过渡也将是 通过K99阶段的培训，尤其是在GPCR样品的表达和纯化方面的培训，促进了这一过程。 科罗拉多大学为开展这项工作提供了世界一流的设施， 在单分子原子力显微镜实验和膜蛋白生物化学的专业知识。