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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。质谱法已成为监测蛋白质折叠各方面（包括动力学）的公认方法。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区域（例如，可能与脂膜相互作用）来检测受保护的蛋白质结构域； (ii) 监测生理 pH 值（快速交换）下 H/D 交换的动力学，然后在低 pH 值（慢速交换）下进行胃蛋白酶消化，并识别受保护区域随时间的变化； (iii) 监测非共价结合，例如某些肽和金属阳离子之间发生的结合。大多数此类实验采用电喷雾质谱法，通常与高效液相色谱分离相结合。 我们正在应用这些技术来了解类固醇生成急性调节蛋白 StAR 的作用模式，该蛋白会增加胆固醇穿过线粒体外膜 (OMM) 的流量，并可能进入线粒体。胆固醇是类固醇合成的重要底物，而阻止这种运输的 StAR 突变可能会导致潜在的致命疾病。 StAR 具有 N 端线粒体前导序列，负责将其导入线粒体，而 N-62 StAR（一种 N 端截短蛋白）具有与野生型相同的类固醇生成活性，尽管它不是导入的。因此，StAR 必须作用于线粒体外膜，而自身不被输入。 3-D 结构尚不清楚，但另一种蛋白质 MLN64（与 StAR 有 37% 同源性）的 StAR 样结构域在碱性条件下形成的晶体显示出富含胆固醇的片状结构。由于质子泵，OMM 中应存在弱酸性环境。使用重组 StAR 的 CD 光谱，我们观察到 pH 3.5 时 _ 螺旋度增加，这表明向熔球的转变。从X射线结构来看，MLN64的C端28个氨基酸形成_螺旋，StAR很可能也是如此。 X射线结构表明，这种螺旋在StAR中对于维持类固醇生成活性至关重要，可以铰链与脂质膜相互作用。如果插入膜中，它可能会引起构象变化以打开结合袋并将胆固醇释放到膜中。 （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。）