Krainer Georg, Gracia Pablo, Frotscher Erik, Hartmann Andreas, Gröger Philip, Keller Sandro, Schlierf Michael
B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany; Molecular Biophysics, University of Kaiserslautern, Kaiserslautern, Germany.
B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany.
Biophys J. 2017 Sep 19;113(6):1280-1289. doi: 10.1016/j.bpj.2017.05.037. Epub 2017 Jun 16.
Structural and dynamic investigations of unfolded proteins are important for understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with other cell components. In the case of outer-membrane proteins (OMPs), unfolded-state properties are of particular physiological relevance, because these proteins remain unfolded for extended periods of time during their biogenesis and rely on interactions with binding partners to support proper folding. Using a combination of ensemble and single-molecule spectroscopy, we have scrutinized the unfolded state of outer-membrane phospholipase A (OmpLA) to provide a detailed view of its structural dynamics on timescales from nanoseconds to milliseconds. We find that even under strongly denaturing conditions and in the absence of residual secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that lack the fast chain-reconfiguration motions expected for an unstructured, fully unfolded chain. The drastically slowed sampling of potentially folding-competent states, as compared with a random-coil polypeptide, may contribute to the slow in vitro folding kinetics observed for many OMPs. In vivo, however, slow intramolecular long-range dynamics might be advantageous for entropically favored binding of unfolded OMPs to chaperones and, by facilitating conformational selection after release from chaperones, for preserving binding-competent conformations before insertion into the outer membrane.
对未折叠蛋白质的结构和动力学研究对于理解蛋白质折叠机制以及未折叠多肽链与其他细胞成分的相互作用至关重要。在外膜蛋白（OMP）的情况下，未折叠状态的特性具有特殊的生理相关性，因为这些蛋白质在生物合成过程中会长时间保持未折叠状态，并依赖与结合伴侣的相互作用来支持正确折叠。通过结合使用系综光谱和单分子光谱，我们仔细研究了外膜磷脂酶A（OmpLA）的未折叠状态，以详细了解其在从纳秒到毫秒的时间尺度上的结构动力学。我们发现，即使在强变性条件下且不存在残留二级结构的情况下，OmpLA也会形成一组缓慢（>100毫秒）相互转换且构象异质的未折叠状态，这些状态缺乏无结构、完全未折叠链所预期的快速链重排运动。与无规卷曲多肽相比，潜在折叠能力状态的采样急剧减慢，这可能导致许多OMP在体外折叠动力学缓慢。然而，在体内，缓慢的分子内长程动力学可能有利于未折叠的OMP与伴侣蛋白进行熵有利的结合，并通过促进从伴侣蛋白释放后的构象选择，在插入外膜之前保持具有结合能力的构象。
