MEMBRANE PROTEIN STRUCTURE AND STABILITY

稳定可溶性球状蛋白三级结构的机制 蛋白质和膜蛋白质被认为是固有不同的。 的 差异是由于缺乏疏水效应， 维持膜蛋白三级结构的力量， 大部分嵌入脂质双层的疏水核心中。 研究 本文提出了一个简单的假设：极性相互作用控制 膜蛋白稳定性 这一假设将用两个 不同的方法：合成肽类似物的结构研究 膜蛋白序列和结构研究 细菌视紫红质及其酶促衍生的肽片段 乳沟 合成肽，模仿一个或多个肽的结构特征， 细菌视紫红质的两个α-螺旋片段，将在 溶液并重构成磷脂囊泡。 这些目标 研究的目的是确定氢键的相对贡献， 与肽-肽或肽内相互作用的离子配对， 二级结构域的相互作用。 将使用的技术 在这些研究中，固有荧光各向异性，圆二色性， 高分辨率1H-NMR、差示扫描量热法和化学分析 交联 细菌视紫红质的研究方向是（1） 区分哪些量热观察到的跃迁来自于 蛋白质-蛋白质相互作用和来自蛋白质内相互作用; （2）在每种相互作用中占主导地位的力是什么。 紫色 膜，重构的单体和寡聚体细菌视紫红质，和 来自酶裂解的细菌视紫红质的重构片段将 通过差示扫描量热法和温度依赖性 可见光谱、圆二色性和荧光光谱 在各种溶液条件下。 这些长期目标 研究的是半合成再生菌紫质的结构 从合成和天然成分。 这一成功将来自于 对造成这种现象的力量的积累的理解 膜蛋白的稳定性。

The mechanisms for stabilizing the tertiary structure of a soluble globular protein and a membrane protein are viewed to be inherently different. The difference arises from the absence of the hydrophobic effect, as a maintaining force in the tertiary structure of membrane proteins that are largely embedded in the hydrophobic core of the lipid bilayer. The studies proposed here follow from a simple hypothesis: Polar interactions control membrane protein stability. This hypothesis will be examined with two different approaches: structural studies on synthetic peptide analogs of membrane protein sequences, and structural studies on intact bacteriorhodopsin and peptide fragments derived from its enzymatic cleavage. Synthetic peptides, that mimic the structural features of one or two Alpha-helical segments of bacteriorhodopsin, will be studied in solution and reconstituted into phospholipid vesicles. The goal of these studies is to determine the relative contribution of hydrogen bonding and ion pairing to peptide-peptide or intra-peptide interactions resulting from the interaction of secondary structural domains. The techniques to be used in these studies are intrinsic fluorescence anisotropy, circular dichroism, high resolution 1H-NMR, differential scanning calorimetry, and chemical crosslinking. Studies on bacteriorhodopsin are directed towards (1) distinguishing which calorimetrically observed transitions are derived from protein-protein interactions and which are from intra-protein interactions; and (2) what forces predominate in each type of interaction. Purple membrane, reconstituted monomeric and oligomeric bacteriorhodopsin, and reconstituted fragments from enzymatically cleaved bacteriorhodopsin will be studied by differential scanning calorimetry and temperature dependent visible spectroscopy, circular dichroism and fluorescence spectroscopy under a variety of solution conditions. The long term goal of these studies is the semi-synthetic regeneration of bacteriorhodopsin structure from both synthetic and native components. This success will result from the accumulated understanding of the forces that contribute to the stability of a membrane protein.