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（TMP）的膜相关信号通路介导的。目前的建议的主要目的是了解的组成和取向的脂质双层（膜）的功能操作的一个原型TMP，G蛋白偶联受体视紫红质，这是主要的蛋白质在视杆外段盘膜感光细胞的影响。这些膜的特征在于具有多不饱和链的高含量的脂质，并且在老化期间经历其组成的变化。不同的脂质组合物对视紫红质构象和功能的生物化学和生物物理后果，它们对将视紫红质掺入膜中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，含有如此高含量的具有多不饱和链的脂质的脂质膜的电化学性质仍然未知。该研究项目的实验方法旨在将视紫红质和选定的视紫红质突变体结合到吸附在固体表面的脂质双层中。我们将使用bicelles纳入视紫红质的变体组成的1，2，-二肉豆蔻酰-sn-甘油-3-磷脂酰胆碱（DMPC），1，2-二己酰-sn-甘油-3-磷酸胆碱（DHPC）和其他脂质在不同的比例。这些模型脂质膜将在固体表面上具有均匀取向的TMP，并代表仿生工作工具。为了确保膜两侧的亲水环境并防止脂质和蛋白质分子与金表面直接接触，将在混合硫代葡萄糖：6-巯基己酸单层上制备浮动脂质膜。双胞的脂质组成将对应于视杆外节椎间盘膜的生理组成。电化学、表面等离子体共振、原位偏振调制红外反射吸收光谱和原子力显微镜实验将被进行以证明模型膜制造的仿生概念。这种最先进的分析方法的协调使用将提供关于膜的宏观性质（电容、电穿孔）、在表面上展开的双胞动力学以及关于模型膜中脂质和视紫红质变体的组成、结构和取向的信息。

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.