STRUCTURAL-BASED MUTAGENESIS STUDY ON THE LOOP REGIONS OF STAR PROTEIN

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。类固醇合成急性调节蛋白(STAR)促进胆固醇快速进入肾上腺和性腺线粒体，在那里胆固醇成为合成所有类固醇激素的第一步的底物。STAR研究出应激反应和生殖所需的线粒体膜。在脂质体存在的情况下，STAR的C末端不能被蛋白质降解，这意味着这个区域与膜有关。与STAR密切相关的两种蛋白质MLN64和StARD4都有晶体结构，表明C-末端有两个环。环的构象变化可能会旋转C末端螺旋，并打开屋顶，使胆固醇与蛋白质结合。两个环中含有带负电荷的残基，而C末端富含带正电荷的残基，这表明盐桥和氢键可能会稳定结构。环周围表面的疏水性表明环可能是脂质装载和卸载的膜对接位置。我们计划确定环区的负电荷残基是否在底物结合和膜结合中发挥作用。首先，我们正在利用密切相关的蛋白质MLN64和StARD4的晶体结构，使用计算机图形实验室开发的“嵌合体”程序，建立一个人类恒星蛋白质模型。从模型中，我们将测量来自环区的带负电荷的残基和来自C-末端的带正电荷的残基之间的侧链距离。然后我们将在硅胶中突变这些残基(S)，并评估它们对蛋白质几何和构象的影响。然后将构建潜在的启发式突变体，并在试验台上进行体内研究。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The steroidogenic acute regulatory protein (StAR) facilitates the rapid movement of cholesterol into adrenal and gonadal mitochondria, where cholesterol becomes the substrate for the first step in the synthesis of all steroid hormones. StAR works out the mitochondrial membrane required for the stress responses and for reproduction. The C-terminus of StAR is resistant to proteolysis in ther presence of liposomes, implying that this region is associated with the membrane. Crystal structures are available for two proteins closely related to StAR, MLN64 and StARD4, revealing that two loops are adjacent to the C-terminus. Conformational changes of the loops could pivot the C-terminal helix and open the roof to allow cholesterol to bind to the protein. The two loops contain negative charged residues, while the C-terminus is rich is positively charged residues, suggesting that salt bridges and hydrogen bonds may stabilize the structure. The hydrophobicity of the surface surrounding the loops suggests that the loop could be the membrane docking site for lipid loading and unloading. We plan to determine whether the negatively charged residues in the loop regions play a role in substrate binding and membrane association. First, are building a human StAR protein model using the crystal structures of the closely related proteins MLN64 and StARD4, using the "Chimera" program developed in the Computer Graphics Lab. From the model, we will measure the side-chain distances between the negatively charged residues from the loop regions and the positively charged residues from C-terminus. We will then mutate these residue(s) in silico and evaluate their effects on the protein geometry and conformation. Potentially heuristic mutants will then be built and studied in vivo at the bench.