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.

项目摘要/摘要 人精氨酸加压素受体2（AVPR2）是收集中表达的螺旋膜蛋白 参与调节尿量的孩子的管道。已知在96个地点的AVPR2突变 引起肾病性糖尿病（NDI），可能是通过促进错误折叠而引起的。加压素拮抗剂药物 （“ vaptans”）已被证明可以挽救细胞表达的表达 本地折叠状态。这是在存在下了解折叠和错误折叠状态的相对能量学 没有配体的缺乏将阐明AVPR2的天然结构动力学以及这些动态如何 通过引起疾病的突变改变。这样的结果将与NDI有关，也将与 由G蛋白偶联受体（GPCR）引起的疾病。但是，已建立的生化技术 化学变性在确定用于测量膜 - 蛋白质热力学稳定性的胶束中 （G）及其在突变或配体结合（G）上的变化受到了几个局限性 不适合研究AVPR2。特别是非本地确定环境，定义不佳 具有明显残留二级结构的变性状态，需要从高变性剂中推断出 浓度导致测得的能量能力不足反映基本的，生物学相关的分子 值。最重要的是，基于化学定义的技术从未成功应用于 GPCRS是因为GPCR在去除变性剂时不会在全球范围内重新分配。这些缺点激发了 该提案的总体目的：开发用于测量膜 - 蛋白能的替代技术，基于膜 - 蛋白质 在力引起的展开而不是化学变性。这样的技术，在原子上实施 力显微镜（AFM）可以研究天然脂质双层中的膜蛋白，并消除 通过一次探测一小部分蛋白质，全球可逆展开。博士后工作 K99相将使用模型膜蛋白菌hopopopin（BR）进一步发展这些基于力的基于力 技术。将实现两个特定的目标：（1）测量BR中点突变的自由能的变化 它的天然双层，没有混淆化学变性剂和（2）数量 BR中的光激活配体异构化。在K99阶段完成这项工作将建立基础 为了独立R00阶段的目的：阐明AVPR2的折叠和配体相互作用能量 使用这些新技术。除了对AVPR2折叠和错误折叠提供具体见解之外， 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充满活力的测量 相关系统，例如AVPR2，而不仅仅是在模型系统中。向独立的过渡也将是 在K99阶段训练促进，最著名的是GPCR样品的表达和纯化。 科罗拉多大学为进行这项工作提供了世界一流的设施，联合会将提供 单分子AFM实验和膜 - 蛋白质生物化学方面的专业知识。