Membrane Protein Folding and Assembly

描述(申请人提供)：截至2003年底，只有75个膜蛋白结构被解析到高分辨率。由于α-螺旋膜蛋白约占基因组序列的30%，从氨基酸序列预测这些蛋白的三维结构是一个重要的目标。这样的预测需要理解两个基本问题：MPS通过SecY/Sec61(转运子)复合体进行生物组装的机制和MPS在其天然脂质双层环境中的物理稳定性原理。该提案的具体目标如下：(1)破译嵌入在膜蛋白氨基酸序列中的“密码”，该“密码”决定多肽片段是否通过Sec61复合体作为跨膜螺旋整合到内质网膜中。这项工作将扩展使用基于将模型蛋白共翻译插入到微生物体中的体外系统获得的初稿的“生物”疏水性标尺。(2)通过对Jannaschii的SecY复合体的分子动力学模拟，深入了解转运子的功能，最近已经确定了该复合体的晶体结构。这些模拟将允许在双层环境中操纵转位子结构，作为了解转位子如何打开和关闭以将TM螺旋释放到双层中的一种手段。(3)建立了一个基于实验的界面疏水性标度，用于描述多肽片段与ER脂形成的双层界面区域之间的相互作用。这一点很重要，因为移位子可能与脂双层结合，区分双层的碳氢化合物核心区域和界面区域。(4)阐明(1)短TM螺旋(-12个氨基酸)和(2)KvAP电压门控K+通道的S4螺旋电压敏感肽的转位辅助插入膜的物理基础。所获得的信息将帮助我们理解如何预测不寻常的膜蛋白的拓扑和结构，例如CIC氯通道。

DESCRIPTION (provided by applicant): Only 75 membrane protein structures had been solved to high resolution by the close of 2003. Because alpha-helical membrane proteins account for ~30% of genome sequences, predicting from amino acid sequence the three-dimensional structures of these proteins is an important goal. Such predictions require understanding two fundamental issues: mechanisms of the biological assembly of MPs by the SecY/Sec61 (translocon) complex and the principles of the physical stability of MPs in their natural lipid bilayer milieu. The specific aims of this proposal address aspects of both issues as follows: (1) Decipher the "code" embedded in membrane protein amino acid sequences that determines whether or not a polypeptide segment is integrated into the endoplasmic reticulum membrane by the Sec61 complex as a transmembrane helix. This work will expand a first draft 'biological' hydrophobicity scale obtained using an in vitro system based upon cotranslational insertion of model proteins into microsomes. (2) Gain insights into translocon function using molecular dynamics simulations of the SecY complex from M. jannaschii, for which the crystallographic structure has been determined recently. These simulations will allow manipulation of the translocon structure in the bilayer environment as a means of learning how the translocon may open and close to release TM helices into the bilayer. (3) Establish an experiment-based interfacial hydrophobicity scale for describing the interactions of polypeptide segments with the interface regions of bilayers formed from ER lipids. This is important because the translocon, presumably in concert with the lipid bilayer, distinguishes between the bilayer's hydrocarbon core and interfacial regions. (4) Clarify the physical basis for translocon-assisted insertion into membranes of (1) short TM helices (-12 amino acids) and (2) a model S4 helix voltage-sensor peptide from the KvAP voltage-gated K+ channel. The information obtained will help us understand how to predict the topology and structure of unusual membrane proteins, such as the CIC chloride channel.