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

项目摘要/摘要 人精氨酸加压素受体2(AVPR2)是一种-螺旋膜蛋白，在集合体中表达 参与调节尿量的肾脏的导管。已知AVPR2基因在约96个位点发生突变 导致肾源性尿崩症(NDI)，可能是通过促进错误折叠。加压素拮抗剂 (“蒸汽蛋白”)被证明可以挽救细胞表面的表达，推测是通过充当伴侣来稳定细胞表面的 原生折叠状态。因此，理解存在时折叠状态和错误折叠状态的相对能量学 而配体的缺失将揭示AVPR2的天然结构动力学以及这些动力学是如何 会因致病突变而改变。这样的结果将与NDI相关，更广泛地说，也与NDI相关 由G蛋白偶联受体(GPCRs)引起的疾病。然而，已建立的生化技术 用于测量膜-蛋白质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)受到几个限制，使其 不适合研究AVPR2。特别是，非本土的洗涤剂环境，定义不佳 变性状态，具有显著的残留二级结构，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、与生物相关的分子 价值观。最重要的是，基于化学变性的技术从未成功地应用于 GPCRs，因为当去掉变性剂时，GPCRs不会全局重新折叠。这些缺点促使 这项建议的总体目标：开发测量膜蛋白能量学的替代技术，基于 力诱导的去折叠，而不是化学变性。这样的技术，在原子上实现 力显微镜(AFM)，可以研究天然脂双层中的膜蛋白，避免了 通过一次探测一小部分蛋白质来全局可逆展开。在博士后期间工作 K99相会利用模型膜蛋白细菌视紫红质(BR)进一步发展这些力基 技巧。将实现两个特定的目标：(1)测量BR的点突变自由能变化 它的天然双层和不混杂的化学变性剂和(2)定量一个 BR中的光激活配体异构化。在K99阶段完成这项工作将奠定基础 对于独立R00相的目的：阐明AVPR2的折叠和配体相互作用的能量学 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外，这篇文章 这项工作将建立一个新的范例，在这个范例中，能量测量可以直接在生物医学中进行 相关系统，如AVPR2，而不仅仅是在模型系统中。向独立的过渡也将是 通过在K99阶段的培训，最显著的是在GPCR样本的表达和纯化方面。 科罗拉多大学为开展这项工作提供了世界级的设施，合作导师将提供 精通单分子原子力显微镜实验和膜蛋白生物化学。

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.