MEMBRANE PROTEINS--STRUCTURE, DYNAMICS AND STABILITY

膜蛋白结构在生物体中的重要性和普遍性在结构数据库中的代表性大大不足。 研究人员建议开发和整合新的液体和固体NMR方法和生物化学方法（二硫键交联和热力学测量），以首次确定大型膜蛋白的结构。 LH1蛋白是光异养真细菌的一种200 kD的异源寡聚膜蛋白，经历了多次可逆的解折叠（解离）和折叠（缔合）反应，非常适合于通过液体和固体NMR进行高分辨率结构测定。 拟议的研究代表了一个重要的方法学概念验证，将鼓励对其他具有直接医学相关性的膜蛋白进行结构研究。液体NMR实验的目的是确定LH 1亚复合物的结构，并确定单个溶液结构可用于构建完整复合物结构的程度。 固体NMR实验将用于确定各种溶剂条件下的亚复合物和完整复合物的结构，目的是确定用于评估膜蛋白折叠（缔合）的NMR特征。液体和固体核磁共振动力学将被确定为侧链和主链运动在各种时间尺度上，他们将比较LH1和它的子复合物。 动力学将与二硫化物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.