Structure-Function Studies of Lipid Binding Proteins

9876575该项目的重点是细胞内脂质结合蛋白家族。这些蛋白质负责细胞内疏水配体的溶解和递送。 脂质结合蛋白结合最积极的脂肪酸和类维生素A，虽然也有一些弱的协会，如血红素和溶血磷脂分子。 尽管一级序列的变异性很大，但由多维NMR和X射线晶体学确定的家族成员的3-D结构是非常重叠的。 所有脂质结合蛋白的标志性特征是β-桶折叠，其形成内部充满水的空腔，该空腔用作配体结合结构域。一旦进入，配体就与外部环境隔离，并沿着结合域的一个壁被刚性地保持沿着，置换约三分之一的空腔水。 尽管在腔内发现了与蛋白质结合的配体，但没有明显的配体进入/退出位点。 脂肪细胞脂质结合蛋白与脂肪酸结合，并用作分析整个脂质结合蛋白类的模型系统。 结构信息的可用性，允许分析有关的几个问题，不仅是蛋白质脂质相互作用的物理基础，但也为配体结合亲和力和选择性的分子分析。 例如，作为配体结合结构域的大的充满水的空腔似乎直观地与蛋白质的目的相矛盾。 如果蛋白质促进水不溶性脂质的溶解，为什么要将它们封闭在充满水的空腔中？ 如果没有开口，配体如何进出空腔？ 控制脂质结合的热力学因素是什么？ 什么决定配体结合亲和力和选择性？ 这些蛋白质是被动的细胞内脂肪酸缓冲剂，还是主动的分子伴侣，指导细胞内脂质的矢量运动？以下假设解决了这些问题：脂肪细胞脂质结合蛋白作为脂肪酸的分子伴侣，促进脂解衍生的脂肪酸从脂肪细胞脂滴到质膜的定向运动。 本项目通过评估一系列旨在解决腔可及性的门突变体的脂肪酸结合过程的动态性质来测试这一假设;以及绘制脂肪细胞敏感性脂肪酶和脂肪细胞脂质结合蛋白之间的接触界面。虽然水溶性化合物可以在生物系统中自由扩散，特定的载体蛋白已经发展用于溶解和递送在水性体系中具有有限溶解度的分子。本研究的重点是在一个家庭的疏水性配体载体蛋白，在细胞内的环境中发挥作用，脂质结合蛋白的结构和功能之间的关系。 正如有载体蛋白在循环系统中起溶解和递送疏水分子的作用（例如，脂蛋白、白蛋白），存在类似的蛋白质家族，其存在于细胞内。 这些蛋白质被称为脂质结合蛋白，负责细胞内疏水配体的溶解和递送。 他们作为可能的监护人的作用将被审查。

9876575The focus of this project is on a family of intracellular lipid binding proteins. These proteins are responsible for the solubilization and delivery of hydrophobic ligands within a cell. The lipid binding proteins bind most avidly to fatty acids and retinoids although some weak association with such molecules as heme and lysophospholipids does occur. Despite the wide variability in primary sequence, the 3-D structure of the family members, as determined by multidimensional NMR and X-ray crystallography, are remarkably superimposable. The signature feature of all the lipid binding proteins is a beta-barrel fold forming an internal, water-filled cavity which serves as the ligand binding domain. Once inside, the ligand is sequestered from the external milieu and is held rigidly along one wall of the binding domain, displacing about one-third of the cavity water. Despite the finding of the ligand bound to the protein within the cavity, there is no obvious site for ligand entry/exit. The adipocyte lipid binding proteins bind to fatty acids and are used as model systems for the analysis of the entire class of lipid binding proteins. The availability of structural information has allowed for the analysis of several questions relating to not only the physical basis for protein lipid interaction, but also for the molecular analysis of ligand binding affinity and selectivity. For example, a large water-filled cavity that serves as the ligand binding domain seems intuitively contradictory to the purpose of the proteins. If the proteins facilitate the solubilization of water-insoluble lipids, why enclose them in a cavity filled with water? How do ligands enter and exit the cavity if there is no opening? What are the thermodynamic factors which govern lipid binding? What determines the ligand binding affinity and selectivity? Do such proteins serve as passive intracellular fatty acid buffers or are they active molecular chaperones, directing the vectoral movement of lipid within cells? The following hypothesis addresses these issues: The adipocyte lipid binding protein functions as a molecular chaperone for fatty acids, facilitating the directed movement of lipolytically derived fatty acids from the adipocyte lipid droplet to the plasma membrane. This project tests this hypothesis by assessing the dynamic nature of the fatty acid binding process with a series of portal mutants designed to address cavity accessibility; and mapping the contact interface between the hormone-sensitive lipase and the adipocyte lipid binding protein.While water soluble compounds may freely diffuse in biological systems, specific carrier proteins have evolved for the solubilization and delivery of molecules which have limited solubility in aqueous systems. The focus of this research is on the relationship between structure and function in one family of hydrophobic ligand carrier proteins which function within the intracellular environment, the lipid binding proteins. Just as there are carrier proteins that function to solubilize and deliver hydrophobic molecules in the circulatory system (e.g., lipoproteins, albumin), there exists an analogous family of proteins that are found intracellularly. These proteins, which have been termed the lipid binding proteins, are responsible for the solubilization and delivery of hydrophobic ligands within a cell. Their role as possible chaperones will be examined.