Machin Jonathan M, Ranson Neil A, Radford Sheena E
Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
Faraday Discuss. 2025 May 8. doi: 10.1039/d4fd00180j.
Biological membranes are asymmetric structures, with asymmetry arising from differences in lipid identity in each leaflet of the bilayer, as well as non-uniform distribution of lipids and small molecules in the membrane. Proteins can also induce and modulate membrane asymmetry based on their shape, sequence and interactions with lipids. How membrane asymmetry affects macromolecular behaviour is poorly understood because of the complexity of natural membrane systems, and difficulties in creating relevant asymmetric bilayer systems . Here, we present a method exploiting the efficient, unidirectional folding of the transmembrane β-barrel outer membrane protein, OmpA, to create asymmetric proteoliposomes with protein-induced dipoles of known direction (arising from sequence variation engineered into the OmpA loops). We then characterise the folding kinetics and stability of different OmpA variants into these proteoliposomes. We find that both the primary sequence of the folding OmpA and the dipole of the membrane into which folding occurs play an important role for modulating the rate of folding. Critically, we find that by complementarily matching the charge on the folding protein to the membrane dipole it is possible to enhance both the folding kinetics and the stability of the folded OmpA. The results hint at how cells might exploit loop charge in membrane-embedded proteins to manipulate membrane environments for adaptation and survival.
生物膜是不对称结构，其不对称性源于双层膜各小叶中脂质特性的差异，以及膜中脂质和小分子的不均匀分布。蛋白质也可根据其形状、序列以及与脂质的相互作用来诱导和调节膜的不对称性。由于天然膜系统的复杂性以及创建相关不对称双层系统的困难，人们对膜不对称性如何影响大分子行为知之甚少。在此，我们提出一种方法，利用跨膜β桶外膜蛋白OmpA的高效单向折叠来创建具有已知方向蛋白质诱导偶极（由工程改造到OmpA环中的序列变异产生）的不对称蛋白脂质体。然后，我们表征不同OmpA变体折叠到这些蛋白脂质体中的折叠动力学和稳定性。我们发现，折叠的OmpA的一级序列以及发生折叠的膜的偶极在调节折叠速率方面都起着重要作用。至关重要的是，我们发现通过使折叠蛋白上的电荷与膜偶极互补匹配，可以提高折叠的OmpA的折叠动力学和稳定性。这些结果暗示了细胞如何利用膜嵌入蛋白中的环电荷来操纵膜环境以实现适应和生存。
