膜蛋白Leptosphaeria rhodopsin二聚化组装的结构机制研究

Leptosphaeria rhodopsin is a eukaryotic and seven helical transmembrane retinal protein. It forms as a stable dimer and may contribute as an important model of membrane protein assembly. We plan to use mainly the site directed electron magnetic resonance technique to identify the helices which contribute to the dimerization, and their relative spatial locations. Meanwhile, we are trying to optimize the crystal quality and hopefully to solve the dimer structure, therefore, directly to identify those residues within the dimer interface. With the structural data in hand, we plan to design new Leptosphaeria rhodopsin mutants, so that we will characterize the dimerization extent by circular dichroism. Furthermore, we will calculate the stability and conformational homogeneity of Leptosphaeria rhodopsin by the light induced denaturation experiment and electron paramagnetic resonance, respectively. Finally, we are trying to illustrate the structural mechanism of the dimerization assembly in Leptosphaeria rhodopsin.

Leptosphaeria rhodopsin是真核生物中发现的一种七次螺旋跨膜的视黄醛蛋白，以特殊稳定的二聚体形式组装，对膜蛋白组装研究具有重要的模型意义。本项目拟从Leptosphaeria Rhodopsin跨膜螺旋结构入手，主要运用空间定位顺磁共振技术，确认参与二聚化组装的螺旋及其相对空间位置；同时进行Leptosphaeria rhodopsin蛋白晶体的培养优化，直接解析出分子间相互作用的螺旋结构与氨基酸残基。根据结构数据进一步设计突变体蛋白，运用圆二色谱等技术表征二聚体组装的程度；以光解实验和顺磁共振分别表征蛋白稳定性与构象均一性。从而初步阐明Leptosphaeria Rhodopsin二聚化组装的结构机制。

本项目主要研究对象是以Leptosphaeria rhodopsin为代表的微生物视紫红质家族蛋白，该蛋白以特殊稳定的二聚体形式组装，对膜蛋白的自发组装研究具有重要的模型意义。本项目主要运用圆二色谱、时间分辨吸收光谱、分子交联技术、顺磁共振、以及晶体衍射等生物物理化学技术和方法，表征Leptosphaeria rhodopsin自发组装过程，分析组装的稳定性结构机制。我们首次提出了二聚体到四聚体的高级组装机制，初步阐明视紫红质蛋白家族中的分子组装的一般性原理。