MEMBRANE PROTEINS--STRUCTURE, DYNAMICS AND STABILITY

由于膜蛋白结构在生物体中的重要性和普遍性，膜蛋白结构在结构数据库中的代表性极低。研究人员建议开发和整合新的液体和固体核磁共振方法和生化方法(二硫键交联和热力学测量)，以首次确定大分子膜蛋白的结构。LH1蛋白是由光异养真核细菌产生的一种200kD的异寡聚体膜蛋白，它经历了多次可逆的去折叠(解离)和折叠(缔合)反应，非常适合于液体和固体核磁共振的高分辨结构测定。拟议的研究代表了一种重要的方法学概念验证，将鼓励对其他具有直接医学意义的膜蛋白进行结构研究。液体核磁共振实验的目标是确定LH1亚复合体的结构，并确定单个溶液结构可以在多大程度上用于构建完整复合体的结构。固体核磁共振实验将用于在各种溶剂条件下确定亚复合体和完整复合体的结构，目的是确定用于评估膜蛋白质折叠(结合)的核磁共振信号。液体和固体的核磁共振动力学将确定侧链和主链在各种时间尺度上的运动，并将它们与LH1及其亚复合体进行比较。动力学将与二硫键交联动力学和缔合热力学有关。总体目标是了解膜蛋白的结构、构象动力学、功能和热力学稳定性之间的关系。

Membrane protein structures are vastly underrepresented in structure databases in relation to their importance and prevalence in living organisms. The investigators propose to develop and integrate novel liquids and solids NMR methods and biochemical methods (disulfide crosslinking and thermodynamic measurements) to determine the structure of a large membrane protein for the first time. LH1 protein, a 200 kD hetero-oligomeric membrane protein from photoheterotrophic eubacteria, undergoes multiple reversible unfolding (dissociation) and folding (association) reactions, and is well-suited for high-resolution structure determination by liquids and solids NMR. The proposed studies represent an important methodological proof-of-concept that will encourage structural investigations on other membrane proteins with direct medical relevance. The goal of the liquids NMR experiments is to determine the structures of LH1 subcomplexes and to ascertain the degree to which the individual solution structures can be used to build up structures of the intact complex. The solids NMR experiments will be used to determine structures of subcomplexes and intact complexes in a variety of solvent conditions, with the goal of determining NMR signatures for assessing membrane protein folding (association). Liquids and solids NMR dynamics will be ascertained for side- chain and backbone motions on a variety of timescales and they will be compared for LH1 and its subcomplexes. The dynamics will be related to the disulfide crosslinking kinetics, and to the thermodynamics of association. The overall goal is to understand the relationship between structure, conformational dynamics, function and thermodynamic stability for membrane proteins.