Structure-Function Studies of Lipid Binding Proteins

9876575本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。脂质结合蛋白与脂肪酸和类维生素a结合最为密切，尽管与血红素和溶血磷脂等分子也存在一些微弱的联系。尽管原生序列有很大的可变性，但由多维核磁共振和x射线晶体学确定的家族成员的三维结构是显著重叠的。所有脂质结合蛋白的特征是一个β -桶状折叠，形成一个内部充满水的空腔，作为配体结合域。一旦进入，配体就与外部环境隔离，并沿着结合域的一面墙固定，取代了大约三分之一的腔水。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出位点。脂肪细胞脂质结合蛋白与脂肪酸结合，并被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几个问题，不仅涉及蛋白质脂质相互作用的物理基础，而且还涉及配体结合亲和力和选择性的分子分析。例如，作为配体结合域的一个大的充满水的空腔在直觉上似乎与蛋白质的目的相矛盾。如果这些蛋白质能促进不溶于水的脂质的溶解，为什么要把它们包裹在一个充满水的空腔里呢？如果没有开口，配体是如何进出腔体的？控制脂质结合的热力学因素是什么？是什么决定了配体的结合亲和力和选择性？这些蛋白质是作为被动的细胞内脂肪酸缓冲液，还是它们是主动的分子伴侣，指导细胞内脂质的矢量运动？下面的假设解决了这些问题：脂肪细胞脂结合蛋白作为脂肪酸的分子伴侣，促进脂聚衍生的脂肪酸从脂肪细胞脂滴定向运动到质膜。该项目通过评估脂肪酸结合过程的动态性质来验证这一假设，该过程具有一系列旨在解决腔体可达性的门脉突变；绘制激素敏感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，但特定的载体蛋白已经进化为在水系统中具有有限溶解度的分子的增溶和递送。本研究的重点是研究一类在细胞内环境中起作用的疏水配体载体蛋白的结构与功能之间的关系，即脂质结合蛋白。正如在循环系统中存在溶解和传递疏水分子的载体蛋白（如脂蛋白、白蛋白）一样，在细胞内也存在类似的蛋白家族。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。他们作为可能的伴侣的角色将被审查。

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.