BIOPHYSICAL STUDIES ON PROTEIN FOLDING BY MASS SPEC:THE STEROIDOGENIC ACUTE REG

通过质谱对蛋白质折叠进行生物物理学研究：类固醇急性调节

• 批准号: 7369035
• 负责人: MICHAEL A BALDWIN
• 金额: $ 2.04万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369035
• 关键词: BIOPHYSICAL; STUDIES; PROTEIN; FOLDING; MASS

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. Mass spectrometry has become an accepted method to monitor aspects of protein folding, including the dynamics. Techniques in common use include: (i) The detection of protected protein domains by limited proteolytic isolation or limited chemical reactivity of regions that, for example, may be interacting with a lipid membrane; (ii) Monitoring the kinetics of H/D exchange at physiological pH (fast exchange) followed by pepsin digestion at low pH (slow exchange) and identification of protected regions as a function of time; and (iii) Monitoring non-covalent associations such as those occurring between some peptides and metal cations. Most such experiments employ electrospray mass spectrometry, often in conjunction with HPLC separations. We are applying these techniques to understand the mode of action of the steroidogenic acute regulatory protein StAR, which increases the flow of cholesterol across the outer mitochondrial membrane (OMM) and perhaps into the mitochondria. Cholesterol is an essential substrate for steroid synthesis, and mutations in StAR that prevent this transport can cause potentially fatal disease. StAR has an N-terminal mitochondrial leader sequence responsible for its import into mitochondria, yet N-62 StAR (an N-terminally truncated protein) has the same steroidogenic activity as wild-type, even though it is not imported. Therefore StAR must act at the outer mitochondrial membrane without itself being imported. The 3-D structure is unknown but crystals formed under alkaline conditions of the StAR-like domain of another protein, MLN64, with 37% homology to StAR, show a _¿sheet rich structure with a pocket for cholesterol. A mildly acidic environment should exist at the OMM due to a proton pump. Using CD spectroscopy of recombinant StAR we observed an increase in _-helicity at pH 3.5 that suggested a transition to a molten globule. From the X-ray structure the C-terminal 28 amino acids of MLN64 form an _-helix, and it is likely that StAR is the same. The X-ray structure suggests this helix, which in StAR is essential to maintain steroidogenic activity, could hinge out to interact with lipid membranes. If inserted into the membrane, it might induce conformational changes to open the binding pocket and release cholesterol into the membrane. (Additional effort and instrument time reported under Collaborative projects and other Technical Research and Development projects.)

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。质谱已经成为一种公认的方法来监测蛋白质折叠的各个方面，包括动力学。常用的技术包括：（i）通过有限的蛋白水解分离或有限的化学反应性检测受保护的蛋白质结构域，例如，可能与脂质膜相互作用的区域;（ii）监测在生理pH下H/D交换的动力学（快速交换），然后在低pH下进行胃蛋白酶消化（慢交换）和鉴定受保护区域作为时间的函数;和（iii）监测非共价缔合，例如在一些肽和金属阳离子之间发生的那些。大多数此类实验采用电喷雾质谱法，通常与HPLC分离结合。 我们正在应用这些技术来了解类固醇生成急性调节蛋白星星的作用模式，该蛋白增加胆固醇穿过线粒体外膜（OMM）并可能进入线粒体的流动。胆固醇是类固醇合成的重要底物，阻止这种转运的星星突变可能导致潜在的致命疾病。星星有一个N-末端线粒体前导序列，负责将其输入线粒体，而N-62星星（一种N-末端截短的蛋白质）具有与野生型相同的类固醇生成活性，即使它不是输入的。因此，星星必须作用于线粒体外膜，而自身不被输入。三维结构是未知的，但在碱性条件下形成的晶体的另一种蛋白质，MLN 64，与星星的同源性为37%的StAR样结构域，显示了一个富含胆固醇口袋的片层结构。由于质子泵，OMM处应存在弱酸性环境。使用重组星星的CD光谱，我们观察到在pH 3.5的_螺旋度的增加，这表明过渡到熔融球。根据X射线结构，MLN 64的C-末端28个氨基酸形成_-螺旋，星星可能也是如此。X射线结构表明，这种螺旋结构在星星中对维持类固醇生成活性至关重要，它可以与脂质膜相互作用。如果插入到膜中，它可能会引起构象变化，打开结合口袋，释放胆固醇到膜中。（在合作项目和其他技术研究与开发项目下报告了额外的工作量和仪器时间。）