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

将 bicelles 组装到浮动的脂跨膜蛋白膜上：一种新的实验方法

• 批准号: 510809665
• 负责人: Privatdozentin Dr. Izabella Brand
• 金额: --
• 依托单位: Institut für Chemie (IfC)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/510809665?language=en
• 关键词: Assembling; bicelles; floating; lipid; transmembrane

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.

重要的生物学反应是由涉及跨膜蛋白（TMP）的膜相关信号通路介导的。当前建议的主要目的是了解脂质双层（膜）的组成和方向的影响，用于典型的TMP的功能操作，G蛋白偶联受体Rhodopsin，这是光多肽细胞中棒外部片段二聚体中的主要蛋白。这些膜的特征是高含量具有多不饱和链的脂质，并在衰老过程中会改变其组成。不同的脂质组成对视紫红质构象和功能具有的生化和生物物理后果，它们对膜中编码的Rhodopsin的影响以及膜品种与Rhodopsin和Rhodopsin突变体的相互相互作用尚不广为人知。此外，含有多不饱和链的脂质含量高的脂质膜的电化学性能仍然未知。该研究项目的实验方法旨在将视紫红质和选定的Rhopopsin突变体掺入脂质双层中，吸附在固体表面上。我们将使用双皮细胞来增加由1,2，-Dimyristoyl-SN-甘油-3-磷脂酰胆碱（DMPC），1,2-二甲酰SN-己酰SN-甘油-3-甘油-3-磷酸胆碱（DHPC）和其他脂质组成的变体。这些模型脂质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。为了确保膜的两侧有亲水性环境，并防止将脂质和蛋白质分子与金表面直接接触，将在混合硫葡萄糖上制备浮动脂质膜：6-甲基己己no糖单层。双丝的脂质组成将对应于杆外段圆盘膜的物理组成。电化学，表面等离子体共振，原位极化调制红外反射分析光谱和原子力显微镜实验将进行，以证明模型膜制造的仿生概念。这种共同使用最先进的分析方法将提供有关膜（电容，电穿孔）的宏观特性，散布在表面上的双核的动力学以及有关模型膜中脂质和Rhopopsin变体的组成，结构和方向的动力学。