Reffay Myriam, Gambin Yann, Benabdelhak Houssain, Phan Gilles, Taulier Nicolas, Ducruix Arnaud, Hodges Robert S, Urbach Wladimir
Laboratoire de Physique Statistique, Ecole Normale Supérieure, UMR 8550 CNRS-UPMC, Université Paris 06, Paris, France.
PLoS One. 2009;4(4):e5035. doi: 10.1371/journal.pone.0005035. Epub 2009 Apr 1.
Membrane proteins are essential in the exchange processes of cells. In spite of great breakthrough in soluble proteins studies, membrane proteins structures, functions and interactions are still a challenge because of the difficulties related to their hydrophobic properties. Most of the experiments are performed with detergent-solubilized membrane proteins. However widely used micellar systems are far from the biological two-dimensions membrane. The development of new biomimetic membrane systems is fundamental to tackle this issue.We present an original approach that combines the Fluorescence Recovery After fringe Pattern Photobleaching technique and the use of a versatile sponge phase that makes it possible to extract crucial informations about interactions between membrane proteins embedded in the bilayers of a sponge phase. The clear advantage lies in the ability to adjust at will the spacing between two adjacent bilayers. When the membranes are far apart, the only possible interactions occur laterally between proteins embedded within the same bilayer, whereas when membranes get closer to each other, interactions between proteins embedded in facing membranes may occur as well.After validating our approach on the streptavidin-biotinylated peptide complex, we study the interactions between two membrane proteins, MexA and OprM, from a Pseudomonas aeruginosa efflux pump. The mode of interaction, the size of the protein complex and its potential stoichiometry are determined. In particular, we demonstrate that: MexA is effectively embedded in the bilayer; MexA and OprM do not interact laterally but can form a complex if they are embedded in opposite bilayers; the population of bound proteins is at its maximum for bilayers separated by a distance of about 200 A, which is the periplasmic thickness of Pseudomonas aeruginosa. We also show that the MexA-OprM association is enhanced when the position and orientation of the protein is restricted by the bilayers. We extract a stoichiometry for the complex that exhibits a strong pH dependance: from 2 to 6 MexA per OprM trimer when the pH decreases from 7.5 to 5.5.Our technique allows to study membrane protein associations in a membrane environment. It provides some challenging information about complexes such as geometry and stoichiometry.
膜蛋白在细胞的交换过程中至关重要。尽管可溶性蛋白研究取得了重大突破，但由于膜蛋白的疏水特性带来的困难，其结构、功能和相互作用仍是一个挑战。大多数实验是用去污剂增溶的膜蛋白进行的。然而，广泛使用的胶束系统与生物二维膜相差甚远。新型仿生膜系统的开发是解决这一问题的基础。我们提出了一种原创方法，该方法结合了条纹图案光漂白后的荧光恢复技术以及使用通用海绵相，这使得提取关于嵌入海绵相双层中的膜蛋白之间相互作用的关键信息成为可能。其明显优势在于能够随意调节两个相邻双层之间的间距。当膜相距较远时，唯一可能的相互作用发生在同一双层内嵌入的蛋白质之间的横向，而当膜彼此靠近时，相对膜中嵌入的蛋白质之间也可能发生相互作用。在用链霉亲和素 - 生物素化肽复合物验证我们的方法后，我们研究了来自铜绿假单胞菌外排泵的两种膜蛋白MexA和OprM之间的相互作用。确定了相互作用模式、蛋白质复合物的大小及其潜在化学计量。特别是，我们证明：MexA有效地嵌入双层中；MexA和OprM不发生横向相互作用，但如果它们嵌入相对的双层中则可以形成复合物；对于相距约200埃（这是铜绿假单胞菌的周质厚度）的双层，结合蛋白的数量达到最大值。我们还表明，当蛋白质的位置和方向受到双层限制时，MexA - OprM的缔合会增强。我们提取了该复合物的化学计量，其显示出强烈的pH依赖性：当pH从7.5降至5.5时，每个OprM三聚体有2至6个MexA。我们的技术允许在膜环境中研究膜蛋白缔合。它提供了有关复合物的一些具有挑战性的信息，如几何形状和化学计量。
