Mechanism of initiation of lipid binding of apolipoprotein A-I

载脂蛋白 A-I 脂质结合的启动机制

• 批准号: 10189632
• 负责人: PAUL Michiel WEERS
• 金额: $ 11.06万
• 依托单位: CALIFORNIA STATE UNIVERSITY LONG BEACH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10189632
• 关键词: Affinity; Amino Acids; Amphipathic Alpha Helix; Antiatherogenic; Apolipoprotein A-I; Apolipoproteins; Atherosclerosis; Binding; Binding Sites; Biological; Biophysics; C-terminal; Cell membrane; Chimera organism; Chimeric Proteins; Cholesterol; Conflict (Psychology); Data; Dissociation; Engineering; Glutamine; Goals; Heart Diseases; High Density Lipoproteins; Human; Hydrophobicity; Individual; Invertebrates; Investigation; Lead; Lecithin; Length; Lipid Binding; Lipids; Liver; Location; Lysine; Modeling; Molecular Conformation; Molecular Sieve Chromatography; Mutagenesis; Mutation; N-terminal; Peripheral; Phospholipids; Plasma; Play; Process; Property; Proteins; Resolution; Role; Side; Site; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Surface; System; Tissues; Transferase; Variant; alpha helix; biophysical analysis; dimer; improved; insight; macrophage; monomer; prevent; receptor binding; reverse cholesterol transport

Project Summary Apolipoprotein (apo) A-I is a multifunctional protein with a well-established role in reverse cholesterol transport and is an important player in heart disease. It is the main protein component of high-density lipoprotein (HDL), which circulates through plasma promoting cholesterol efflux. While a high-resolution structure is not known yet, extensive biophysical analysis has suggested that the 28 kDa protein is made of two domains, each of which contain amphipathic α-helices for association with lipid surfaces. The C-terminal (CT) domain contains helical segments that initiate lipid binding, and is also the site responsible for self-association. It is a critical part of the protein needed for maturation of lipid-free apoA-I into HDL. Conflicting data exist about the role of the N-terminal (NT) helices in this process, as well as the precise helical segments of the CT domain. We have recently discovered that CT lysine residues are critical for self-association, and were able to create a monomeric version of the protein. To identify apoA-I helical segments important for initiation of lipid binding and self-association, which are closely connected, we developed a chimeric protein. This chimera will be used to identify which helical segments of apoA-I, both NT and CT α-helices, are required for initiation of lipid binding and self-association. To identify the specific amino acid residues of the CT domain required in self-association, site-directed mutagenesis will be employed. All proteins will be expressed in a bacterial expression system, purified by affinity and size-exclusion chromatography, and characterized for structure and function. The results of this study will lead to a much better understanding in the domain organization of this critical apolipoprotein, their structure function relationship, and may also provide opportunities for high-resolution structural analysis using monomeric apoA-I.

项目摘要 载脂蛋白（APO）A-I是一种多功能蛋白 胆固醇的运输是心脏病的重要参与者。它是主要蛋白 高密度脂蛋白（HDL）的成分，该脂蛋白（HDL）通过血浆促进的圆圈 胆固醇外排。虽然尚不清楚高分辨率结构，但广泛的生物物理 分析表明28 kDa蛋白是由两个域组成的，每个结构域都包含 与脂质表面关联的两亲性α-螺旋。 C末端（CT）域包含 启动脂质结合的螺旋段，也是负责自我关联的部位。它 是将无脂质apoA-I成熟到HDL中所需的蛋白质的关键部分。冲突 存在有关N末端（NT）螺旋在此过程中的作用的数据，以及精度 CT域的螺旋段。我们最近发现CT赖氨酸残留物是 对于自我关联至关重要，并且能够创建蛋白质的单体版本。到 识别apoa-i螺旋段对于脂质结合和自我关联的主动性很重要， 密切相关的，我们开发了一种嵌合蛋白。这个嵌合体将习惯 确定启动的ApoA-I的螺旋段，NT和CTα-螺旋。 脂质结合和自我关联。确定CT结构域的特定氨基酸保留 自我关联所需的需要，将雇用位置定向的诱变。所有蛋白质将是 在细菌表达系统中表达，通过亲和力和尺寸排斥纯化 色谱法，并以结构和功能为特征。这项研究的结果将领导 在这种关键的apolipopprotein的领域组织中更好地理解他们 结构功能关系，也可能为高分辨率结构提供机会 使用单体ApoA-I分析。