Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds, UK.
Nat Chem. 2023 Dec;15(12):1754-1764. doi: 10.1038/s41557-023-01319-6. Epub 2023 Sep 14.
Biological membranes consist of two leaflets of phospholipid molecules that form a bilayer, each leaflet comprising a distinct lipid composition. This asymmetry is created and maintained in vivo by dedicated biochemical pathways, but difficulties in creating stable asymmetric membranes in vitro have restricted our understanding of how bilayer asymmetry modulates the folding, stability and function of membrane proteins. In this study, we used cyclodextrin-mediated lipid exchange to generate liposomes with asymmetric bilayers and characterize the stability and folding kinetics of two bacterial outer membrane proteins (OMPs), OmpA and BamA. We found that excess negative charge in the outer leaflet of a liposome impedes their insertion and folding, while excess negative charge in the inner leaflet accelerates their folding relative to symmetric liposomes with the same membrane composition. Using molecular dynamics, mutational analysis and bioinformatics, we identified a positively charged patch critical for folding and stability. These results rationalize the well-known 'positive-outside' rule of OMPs and suggest insights into the mechanisms that drive OMP folding and assembly in vitro and in vivo.
生物膜由两层磷脂分子组成，形成双层膜，每个分子层包含独特的脂质组成。这种不对称性是由专门的生化途径在体内产生和维持的，但在体外创建稳定的不对称膜的困难限制了我们对双层不对称如何调节膜蛋白的折叠、稳定性和功能的理解。在这项研究中，我们使用环糊精介导的脂质交换生成具有不对称双层的脂质体，并表征两种细菌外膜蛋白（OMP），OmpA 和 BamA 的稳定性和折叠动力学。我们发现脂质体外层的过量负电荷会阻碍它们的插入和折叠，而内层的过量负电荷会加速它们的折叠，与具有相同膜组成的对称脂质体相比。使用分子动力学、突变分析和生物信息学，我们确定了一个对折叠和稳定性至关重要的正电荷补丁。这些结果合理地解释了 OMP 众所周知的“正向外”规则，并为体外和体内驱动 OMP 折叠和组装的机制提供了见解。
