Lipoprotein Structure and Apoprotein Conformation

脂蛋白结构和脱辅基蛋白构象

• 批准号: 7140007
• 负责人: DAVID ATKINSON
• 金额: $ 44.56万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140007
• 关键词: X ray crystallography; apolipoproteins; blood lipoprotein; cardiovascular disorder; chemical models; conformation; cryoelectron microscopy; high density lipoproteins; human tissue; lipid structure; low density lipoprotein; low density lipoprotein receptor; point mutation; protein structure function; structural biology; synthetic peptide; thermodynamics; three dimensional imaging /topography

Our long-term objectives remain to elucidate in molecular detail the structure, stability and dynamic properties of the plasma lipoproteins and apolipoproteins that are vital to an understanding of lipid interactions, apoprotein exchange, lipoprotein-cell-surface interactions, receptor-mediated lipoprotein uptake, and lipoprotein inter-conversions. The conformational adaptability of the exchangeable apoproteins is essential to both their structural role in lipoprotein stabilization and their functional roles as cofactors for enzymes, ligands for receptors, or mediators of reverse cholesterol transport. A detailed understanding of apoprotein structural stability and adaptability is vital to further progress in understanding lipoprotein structure and function. The precise molecular mechanisms of this unique structural adaptability remain unclear, and remain the focus of the proposed research. Continuing our previous work, we will focus on structural investigations of lipoproteins (HDL and LDL) and apolipoproteins to highest possible resolution, using state-of-the-art methods of molecular biophysics and structural biology. The structure and stabilizing interactions of synthetic or expressed peptides that model important structural and functional units in the sequences of the exchangeable apoproteins (primarily apoA-l) will be determined. These peptides are designed based on the 11/22 residue helical segments comprising native apoA-l, and on an "idealized" consensus sequence for the fundamental 11/22-mer tandem repeat in the sequence of the exchangeable apoproteins. Structural and thermodynamic studies of mutant forms of apoA-l encompassing point and deletion mutants will concentrate on the role of specific regions of the apoA-l molecule in its conformation and stability. The three-dimensional structure of intact LDL, with emphasis on the topology and the molecular conformation of the apo-B100 at the lipoprotein surface will be determined by cryo-electron microscopy and 3D-image reconstruction. The focus will be on the organization of apo-B and the localization of structural and functional domains on the LDL particle, using a combination of site-specific immuno-labeling and direct visualization of the bound LDL receptor. The structure of the LDL receptor domains with and without bound apoE and/or apoB peptides will be determined using crystallographic methods. This should ultimately lead to understanding the molecular mechanisms underlying the physiological functions of plasma lipoproteins and lead to the development for molecular approaches to the prevention of heat disease and atherosclerosis.

我们的长期目标仍然是在分子细节上阐明结构、稳定性和动力学 对理解脂质至关重要的血浆脂蛋白和载脂蛋白的性质 相互作用，载脂蛋白交换，脂蛋白-细胞-表面相互作用，受体介导的脂蛋白摄取， 和脂蛋白相互转化。可交换的脱辅基蛋白的构象适应性为 对它们在脂蛋白稳定中的结构作用和作为辅助因子的功能作用都是必不可少的 酶、受体的配体或胆固醇反向运输的介体。详细了解 载脂蛋白结构的稳定性和适应性是进一步理解脂蛋白的关键 结构和功能。这种独特的结构适应性的确切分子机制仍然存在 不清楚，并仍然是拟议研究的重点。继续我们之前的工作，我们将重点放在 尽可能高分辨率的脂蛋白(高密度脂蛋白和低密度脂蛋白)和载脂蛋白的结构研究， 使用最先进的分子生物物理学和结构生物学方法。结构与稳定化 人工合成或表达的多肽的相互作用，这些多肽模拟了细胞内重要的结构和功能单位 可交换的载脂蛋白(主要是载脂蛋白A-L)的序列将被确定。这些多肽是 基于含有天然载脂蛋白A-L的11/22残基螺旋片段而设计，并基于一种“理想化” 基本11/22-聚体串联重复序列在可交换序列中的一致序列 载脂蛋白。载脂蛋白A-L包含点和突变型的结构和热力学研究 缺失突变体将集中在载脂蛋白A-L分子的特定区域在其构象中的作用 和稳定性。完整低密度脂蛋白的三维结构，重点是拓扑结构和分子结构 载脂蛋白B100在脂蛋白表面的构象将通过冷冻电子显微镜和 三维图像重建。重点将放在载脂蛋白B的组织和结构和结构的本地化上 结合使用位点特异性免疫标记和直接 结合的低密度脂蛋白受体的可视化。有结合和无结合的低密度脂蛋白受体结构 ApoE和/或apoB多肽将使用结晶学方法进行测定。这最终应该会导致 了解血浆脂蛋白生理功能的分子机制 导致分子方法的发展，以预防中暑和动脉粥样硬化。