Assembling bicelles to floating lipid- transmembrane protein membranes: A novel experimental approach

重要的生物反应是由涉及跨膜蛋白的膜相关信号通路介导的。本研究的主要目的是了解脂质双分子层（膜）的组成和取向对典型TMP的功能运作的影响，即G蛋白偶联受体视紫红质，它是光感受器细胞中杆外节盘膜中的显性蛋白。这些膜的特点是具有多不饱和链的脂质含量高，并且在老化过程中其组成发生变化。不同的脂质组成对视紫红质构象和功能的生物化学和生物物理影响，它们对视紫红质在膜中的结合的影响，以及膜品种与视紫红质和视紫红质突变体的相互作用尚不清楚。此外，含有如此高含量的脂质和多不饱和链的脂质膜的电化学性质仍然未知。本研究项目的实验方法旨在将视紫红质和选择的视紫红质突变体结合到固体表面吸附的脂质双层中。我们将使用双筒体纳入由1,2，-二肉豆醇-sn-甘油-3-磷脂酰胆碱（DMPC）， 1,2-二己醇-sn-甘油-3-磷脂酰胆碱（DHPC）和其他不同比例的脂质的视紫红质变体。这些模型脂膜将在固体表面上具有均匀定向的TMP，并代表仿生工作工具。为了保证膜两侧的亲水环境，防止脂质和蛋白质分子与金表面直接接触，在混合巯基葡萄糖：6-巯基己酸单层上制备浮动脂质膜。细胞的脂质组成与杆外节盘膜的生理组成相对应。将进行电化学、表面等离子体共振、原位极化调制红外反射吸收光谱和原子力显微镜实验来验证模型膜制造的仿生概念。这种最先进的分析方法的协同使用将提供有关膜的宏观特性（电容，电穿孔），在表面上扩散的小细胞动力学，以及模型膜中脂质和视紫红质变体的组成，结构和方向的信息。

Important biological responses are mediated by membrane–associated signaling pathways involving transmembrane proteins (TMP). The main objective of the current proposal is to understand the influence of the composition and orientation of the lipid bilayer (membrane) for the functional operation of a prototypical TMP, the G protein-coupled receptor rhodopsin, which is the dominant protein in rod outer segment disc membranes in photoreceptor cells. These membranes are characterized by a high content of lipids with polyunsaturated chains and undergo a change of their composition during aging. The biochemical and biophysical consequences that different lipid compositions have on rhodopsin conformation and function, their impact on incorporating rhodopsin in membranes and the mutual interactions of membrane varieties with rhodopsin and rhodopsin mutants are not well known yet. Further, the electrochemical properties of lipid membranes containing such high content of lipids with polyunsaturated chains are still unknown. The experimental approach of the research project aims at incorporating rhodopsin and selected mutants of rhodopsin into lipid bilayers adsorbed on solid surfaces. We will use bicelles for incorporating rhodopsin variants consisting of 1,2,-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) and other lipids at different ratios. These model lipid membranes will have a uniformly oriented TMP on a solid surface and represent a biomimetic working tool. To ensure a hydrophilic environment on both sides of the membrane and prevent a direct contact of the lipid and protein molecules with the gold surface, floating lipid membranes will be prepared on the mixed thioglucose:6-mercaptohexanoic monolayer. The lipid composition of bicelles will correspond to the physiological composition of rod outer segment disc membranes. Electrochemical, surface plasmon resonance, in situ polarization modulation infrared reflection absorption spectroscopy and atomic force microscopy experiments will be carried out to proof the biomimetic concept of model membrane fabrication. This concerted use of state of the art analytical methods will provide information about the macroscopic properties of the membrane (capacitance, electroporation), kinetics of bicelles spreading on surfaces, and about the composition, structure and orientation of lipids and rhodopsin variants in model membranes.