Analysis of a Conformational Switch

构象开关分析

• 批准号: 7015793
• 负责人: LINDA M ROBERTS
• 金额: $ 0.4万
• 依托单位: CALIFORNIA STATE UNIVERSITY SACRAMENTO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7015793
• 关键词: animal genetic material tag; antiatherogenic agent; apolipoproteins; chemical binding; circular dichroism; conformation; fluorescence spectrometry; gene deletion mutation; high density lipoproteins; human genetic material tag; lipids; protein purification; protein structure; protein structure function; proteolysis; site directed mutagenesis; spectrometry; structural biology; zebrafish

DESCRIPTION (provided by applicant): Apolipoprotein A-I (apo A-I), which binds discoidal and spherical lipid surfaces via a series of 11- and 22-mer amphipathic helices, is the major protein constituent of high density lipoprotein (HDL). Apo A-I has antiatherogenic properties, which include reverse cholesterol transport and inhibition of LDL oxidation. The molecular mechanisms governing these and other apo A-I functions are largely unknown. Previous work demonstrating proteolytic susceptibility near residue 42 in lipid-bound but not lipid-free apo A-I stimulated the design of a deletion mutant, delta 1-43 apo A-I. Removal of residues 1-43 resulted in conversion of the lipidfree protein structure to the lipid-bound conformation, even in the absence of lipid, suggesting that this region acts as a conformational switch between lipid-free and lipid-bound protein conformations. The crystal structure of delta 1-43 apo A-I (the only crystal structure of apo A-I to date) provided critical information concerning the disposition of amphipathic helices on lipid surfaces. However, it provides no information concerning the structure of residues 1-43, which have been proposed to form a globular domain. Knowledge of the structure in this region of the protein is critical for understanding apo A-I's anti-atherogenic properties as well as for understanding mechanisms governing the propensity for amino-terminal mutants of apo A-I to form amyloids. Our research aims to fill the gap in knowledge of the 1-43 region through two Specific Aims: we will delineate the portion of 1-43 needed to maintain the protein in its lipid-free state by analyzing the structures of proteins with smaller deletions within the 1-43 region (Specific Aim 1), and evaluate the conservation of the conformational switch properties of the 1-43 region by comparison of full-length and delta 1-43 analogs of zebrafish apo A-I (Specific Aim 2). The structures and lipid-binding properties of the proposed mutants will be evaluated using spectroscopic and physico-chemical techniques that have been shown to distinguish the normal lipid-free structure from that of the delta 1-43 protein including circular dichroism and fluorescence spectroscopy, limited proteolysis, reconstituted HDL formation, and disassembly of multilamellar vesicles.

描述（由申请人提供）：载脂蛋白A-I（Apo A-I），通过一系列11-和22-mer两亲性螺旋结合盘状和球形脂质表面，是高密度脂蛋白（HDL）的主要蛋白质。 Apo A-I具有抗动脉粥样硬化特性，其中包括反向胆固醇转运和LDL氧化的抑制。 控制这些apo A-I功能的分子机制在很大程度上尚不清楚。 先前的工作表明，在脂质结合但不含脂质的APO A-I中残基42附近的蛋白水解易感性刺激了缺失突变体Delta 1-43 Apo A-I的设计。 去除残基1-43导致脂质蛋白结构的转化为脂质结合的构象，即使在没有脂质的情况下，也表明该区域充当无脂质和脂质结合蛋白构象之间的构象转换。 Delta 1-43 Apo A-I的晶体结构（到目前为止，APO A-I的唯一晶体结构）提供了有关在脂质表面上处置两亲螺旋螺旋的关键信息。 但是，它没有提供有关残基1-43结构的信息，这些信息已提议形成一个球状结构域。 对蛋白质区域的结构的了解对于理解Apo A-I的抗动脉粥样硬化特性以及理解管理Apo A-I氨基末端突变体形成淀粉样蛋白的倾向的机制至关重要。 我们的研究旨在通过两个具体目的来填补1-43区域的知识空白：我们将通过分析1-43区域内具有较小缺失的蛋白质的结构来描述将蛋白质保持在其无脂质状态的1-43的部分，并根据1-43区域中的缺失较小（特定的目标1），并评估1-43区域的构型开关属性的保护属性，即1-43区域的构型构造属性 - 43区域的构造属性 - 43区域的构型 - 43区域。斑马鱼Apo A-I的类似物（特定目标2）。 将使用光谱和物理化化学技术评估所提出的突变体的结构和脂质结合特性，这些技术已证明，这些技术将正常的无脂质结构与三角洲1-43蛋白的结构区分开来，包括圆形二色症和荧光光谱，荧光光谱，有限的蛋白质分析，量子分解，均匀的形式，以及综合形式，以及杂物化的形式，以及均匀的形式，以及均匀的形式。