MEMBRANE PROTEINS--STRUCTURE, DYNAMICS AND STABILITY

膜蛋白结构在结构数据库中相对于它们在生物体中的重要性和流行程度，被大大低估了。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蛋白是一种来自光异养真细菌的200 kD异寡聚膜蛋白，经过多次可逆的展开（解离）和折叠（结合）反应，非常适合于液体和固体核磁共振的高分辨率结构测定。拟议的研究代表了一个重要的方法学概念验证，将鼓励其他膜蛋白的结构研究与直接医学相关。液体核磁共振实验的目的是确定LH1亚配合物的结构，并确定单个溶液结构可用于构建完整配合物结构的程度。固体核磁共振实验将用于确定亚配合物和完整配合物在各种溶剂条件下的结构，目的是确定评估膜蛋白折叠（关联）的核磁共振特征。液体和固体核磁共振动力学将确定在各种时间尺度上的侧链和主链运动，并将对LH1及其亚配合物进行比较。动力学将与二硫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.