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.)

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。质谱已成为监测蛋白质折叠方面的一种接受方法，包括动力学。常用技术包括：（i）通过有限的蛋白水解隔离或有限的区域化学反应性检测受保护的蛋白质结构域，例如，这些区域可能与脂质膜相互作用； （ii）监测物理pH（快速交换）的H/D交换的动力学，然后在低pH（缓慢交换）处进行胃蛋白酶消化，并鉴定受保护区域作为时间的函数； （iii）监测非共价关联​​，例如某些胡椒粉和金属阳离子之间发生的关联。大多数这样的实验员工电喷雾质谱法通常与HPLC分离结合使用。我们正在应用这些技术来了解类固醇急性调节蛋白星的作用方式，从而增加了胆固醇在线粒体外膜（OMM）以及可能进入线粒体的流动。胆固醇是类固醇合成的必不可少的底物，而恒星中的突变可防止这种运输可能导致潜在的致命疾病。 StAR具有负责其进口到线粒体的N端线粒体领导者的序列，但是N-62 Star（N末端截断的蛋白质）具有与野生型相同的类固醇活性活性，即使它没有进口。因此，星必须在线粒体外膜上作用，而不会自身进口。 3-D结构是未知的，但是在另一种蛋白质MLN64的星状结构域的酒精条件下形成的晶体，具有37％的同源性，显示了富含薄片的结构，带有一个用于胆固醇的口袋。由于质子泵，应在OMM处存在一个轻度的酸性环境。使用重组星的CD光谱法，我们观察到pH 3.5时_螺旋的增加，这表明向熔融球发生了过渡。从X射线结构中，MLN64的C末端28氨基酸形成_-螺旋，并且恒星很可能是相同的。 X射线结构表明，在恒星中，这对于维持类固醇活性至关重要，可以铰链与脂质膜相互作用。如果插入膜，它可能会引起宪法变化，以打开结合口袋并将胆固醇释放到膜中。 （在协作项目和其他技术研发项目下报告的额外努力和仪器时间。）