MEMBRANE PROTEIN STRUCTURE AND STABILITY

稳定可溶性球状分子三级结构的机制 蛋白质和膜蛋白被认为是本质上不同的。这个 差异产生于疏水效应的缺失，如 膜蛋白三级结构中的维持力 主要嵌入在脂双层的疏水核心中。这些研究 这里提出的建议源于一个简单的假设：极地相互作用控制 膜蛋白稳定性。我们将用两个例子来检验这一假设 不同方法：合成肽类似物的结构研究 膜蛋白序列和完整的结构研究 细菌视紫红质及其酶衍生的多肽片段 乳沟。合成肽，模仿一种或一种的结构特征 细菌视紫红质的两个α-螺旋片段将在 溶解并重组成磷脂小泡。这些项目的目标是 研究的目的是确定氢键和氢键的相对贡献 离子配对导致的多肽-多肽或多肽内相互作用 二级结构域之间的相互作用。要使用的技术 在这些研究中有本征荧光各向异性、圆二向色性、 高分辨~1H-核磁共振、差示扫描量热法和化学 交联剂。细菌视紫红质的研究方向是(1) 区分哪些量热学观察到的转变源自 蛋白质-蛋白质相互作用和蛋白质内相互作用； 以及(2)在每种类型的互动中，什么力量占主导地位。紫色 膜、重组单体和寡聚细菌视紫红质，以及 经酶切的细菌视紫红质重组片段将 用差示扫描量热法和温度依赖性研究 可见光谱、圆二色谱和荧光光谱 在各种溶液条件下。这些项目的长期目标是 细菌视紫红质结构的半合成再生研究 来自合成成分和天然成分。这一成功将产生于 对促成经济衰退的各种力量的累积理解 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.