Huang S, Murphy S, Matouschek A
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208-3500, USA.
Proc Natl Acad Sci U S A. 2000 Nov 21;97(24):12991-6. doi: 10.1073/pnas.230243097.
Many biological processes require proteins to undergo conformational changes at the surface of membranes. For example, some precursor proteins unfold at the surface of mitochondria and chloroplasts before translocation into the organelles, and toxins such as colicin A unfold to the molten globule state at bacterial surfaces before inserting into the cell membrane. It is commonly thought that the membrane surfaces and the associated protein machinery destabilize the substrate proteins and that this effect is required for membrane insertion or translocation. One of the best characterized translocation processes is protein import into mitochondria. By measuring the contributions of individual interactions within a model protein to its stability at the mitochondrial surface and in free solution, we show here that the mitochondrial surface neither induces the molten globule state in this protein nor preferentially destabilizes any type of interaction (e.g., hydrogen bonds, nonpolar, etc.) within the protein. Because it is not possible to measure absolute protein stability at the surface of mitochondria, we determined the stability of a tightly associated protein-protein complex at the mitochondrial import site as a model of the stability of a protein. We found the binding constants of the protein-protein complex at the mitochondrial surface and in free solution to be identical. Our results demonstrate that the mitochondrial surface does not destabilize importing precursor proteins in its vicinity.
许多生物过程需要蛋白质在膜表面发生构象变化。例如，一些前体蛋白在转运到线粒体和叶绿体之前，会在线粒体和叶绿体表面展开，而诸如大肠杆菌素A等毒素在插入细胞膜之前，会在细菌表面展开成熔球状态。人们普遍认为，膜表面及相关的蛋白质机制会使底物蛋白不稳定，且这种效应是膜插入或转运所必需的。研究得最为透彻的转运过程之一是蛋白质导入线粒体。通过测量模型蛋白中各个相互作用对其在线粒体表面和游离溶液中稳定性的贡献，我们在此表明，线粒体表面既不会诱导该蛋白形成熔球状态，也不会优先破坏该蛋白内任何类型的相互作用（例如氢键、非极性相互作用等）。由于无法测量蛋白质在线粒体表面的绝对稳定性，我们测定了线粒体导入位点处紧密结合的蛋白质 - 蛋白质复合物的稳定性，以此作为蛋白质稳定性的模型。我们发现该蛋白质 - 蛋白质复合物在线粒体表面和游离溶液中的结合常数是相同的。我们的结果表明，线粒体表面不会使其附近正在导入的前体蛋白不稳定。
