Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
Biophys J. 2011 May 4;100(9):2131-40. doi: 10.1016/j.bpj.2011.03.025.
The spontaneous folding of two Neisseria outer membrane proteins, opacity-associated (Opa)(60) and Opa(50) into lipid vesicles was investigated by systematically varying bulk and membrane properties. Centrifugal fractionation coupled with sodium dodecyl sulfate polyacrylamide gel electrophoresis mobility assays enabled the discrimination of aggregate, unfolded membrane-associated, and folded membrane-inserted protein states as well as the influence of pH, ionic strength, membrane surface potential, lipid saturation, and urea on each. Protein aggregation was reduced with increasing lipid chain length, basic pH, low salt, the incorporation of negatively charged guest lipids, or by the addition of urea to the folding reaction. Insertion from the membrane-associated form was improved in shorter chain lipids, with more basic pH and low ionic strength; it is hindered by unsaturated or ether-linked lipids. The isolation of the physical determinants of insertion suggests that the membrane surface and dipole potentials are driving forces for outer membrane protein insertion and folding into lipid bilayers.
我们系统性地改变了本体和膜性质，研究了两种脑膜炎奈瑟菌外膜蛋白（Opa）（60）和 Opa（50）自发折叠到脂质体中的情况。离心分离与十二烷基硫酸钠聚丙烯酰胺凝胶电泳迁移率分析相结合，能够区分聚集体、未折叠的膜相关和折叠的膜插入蛋白状态，以及 pH 值、离子强度、膜表面电势、脂质饱和度和脲对每种状态的影响。随着脂质链长的增加、碱性 pH 值、低盐、带负电荷的客体脂质的掺入或向折叠反应中添加脲，蛋白聚集减少。在较短链脂质中，从膜相关形式的插入得到改善，碱性 pH 值和低盐度；它受到不饱和或醚键脂质的阻碍。插入物理决定因素的分离表明，膜表面和偶极电势是推动外膜蛋白插入和折叠到脂质双层中的驱动力。
