Membrane curvature sensors and inducers studied by site-directed spin labeling

Unrestricted Control and regulation of membrane curvature play important roles in membrane trafficking and remodeling events. These processes are mediated by proteins that can sense and/or induce membrane curvature. The focus of my thesis is to understand the underlying molecular mechanisms that enable proteins to remodel membranes. Structural and biophysical studies were performed on the curvature-inducing proteins epsin (an ENTH protein) and endophilin (a BAR protein), both involved in membrane remodeling during endocytosis. Membrane interaction of α-synuclein, a curvature sensor, was also studied.; According to EPR and site-directed spin labeling, α-synuclein takes up an elongated helix with a helical periodicity of 11 amino acids per 3 turns (α11/3) in the membrane-bound form. Combining EPR and structural refinement, we found that the extended helical structure in the presence of membranes has a superhelical twist. This may be a result of α11/3 and allows the protein to have an elongated helical structure. The extended helix is at the level of the phosphate headgroup where it likely compensates for the curvature strain that is present in highly curved vesicles.; BAR domains are found among proteins involved in endocytosis and represents a membrane-binding and curvature-inducing module. The crystal structure of the BAR domain-containing protein endophilin show that it is a banana-shaped dimer composed of a 6-helical bundle arranged in a coiled-coil and suggests a scaffolding mechanism for the sensing and generation of membrane curvature. We found that many structural features of the crystal structure dimer are retained upon membrane interaction. The data also suggest that the BAR domain is at a distance from phosphate level of the membrane and is more likely to interact with the outermost region of the headgroup. The two regions not resolved in the crystal structure undergo a conformational change to a helical structure in the presence of membrane. The centers of both helices are at the level of the phosphate headgroup, where they are likely to promote membrane curvature by wedging lipids apart. Membrane-bound epsin was also analyzed.; Our work shows the importance of amphipathic helices. These studies will assist in the elucidation of the mechanism of membrane curvature regulation.膜曲率的无约束调控在膜运输与重塑事件中发挥重要作用，此类过程由可感知并/或诱导膜曲率的蛋白质所介导。本论文的研究重点是解析蛋白质介导膜重塑的潜在分子机制。我们针对两种在胞吞过程中参与膜重塑的曲率诱导蛋白——epsin（ENTH蛋白）与endophilin（BAR蛋白）开展了结构与生物物理研究；同时还研究了作为曲率感应蛋白的α-突触核蛋白（α-synuclein）与膜的相互作用。 基于电子顺磁共振（EPR, Electron Paramagnetic Resonance）与定点自旋标记（Site-Directed Spin Labeling）技术的分析结果，膜结合状态下的α-突触核蛋白呈现伸长螺旋构象，其螺旋周期为每3圈包含11个氨基酸残基（α11/3）。结合EPR技术与结构精修手段，我们发现膜环境下该蛋白的伸长螺旋结构存在超螺旋扭转。这一现象或源于α11/3螺旋周期，使得该蛋白能够维持伸长螺旋构象。该伸长螺旋定位于磷脂头部基团层面，或可补偿高曲率囊泡中存在的曲率张力。 BAR结构域（BAR domain）广泛存在于参与胞吞过程的蛋白质中，是一类兼具膜结合与曲率诱导功能的结构模块。含BAR结构域的endophilin的晶体结构（crystal structure）显示，其为香蕉形二聚体，由6个螺旋束组成卷曲螺旋（coiled-coil）结构，这为膜曲率的感应与生成提供了支架机制。我们发现，该晶体结构中二聚体的诸多结构特征在膜结合状态下得以保留。实验数据同时表明，BAR结构域与膜的磷酸头部基团存在一定距离，更倾向于与头部基团的最外层区域相互作用。晶体结构中未解析的两个区域在膜环境下发生构象转变，形成螺旋结构。这两个螺旋的中心均位于磷脂头部基团层面，可能通过将脂质推开以促进膜曲率的形成。我们还对膜结合状态下的epsin进行了分析。 本研究凸显了两亲性螺旋（amphipathic helices）的重要性。上述研究将有助于阐明膜曲率调控的分子机制。