Membrane Protein Folding and Assembly

描述（由申请人提供）：截至2003年底，仅有75个膜蛋白结构得到了高分辨率的解析。由于α -螺旋膜蛋白占基因组序列的约30%，因此从氨基酸序列中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（转位体）复合物的生物组装机制和MPs在其天然脂质双分子层环境中的物理稳定性原理。本提案的具体目标涉及以下两个方面：(1)破译嵌入在膜蛋白氨基酸序列中的“密码”，该“密码”决定了多肽片段是否被Sec61复合体作为跨膜螺旋整合到内质网膜中。这项工作将扩大使用基于共翻译插入模型蛋白到微粒体的体外系统获得的“生物”疏水性规模的初稿。(2)利用M. jannaschii的SecY复合体的分子动力学模拟来深入了解转座功能，该复合体的晶体结构最近已经确定。这些模拟将允许在双层环境中操纵易位子结构，作为学习易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)建立基于实验的界面疏水性尺度，描述多肽片段与内质网双层界面区域的相互作用。这很重要，因为转座子可能与脂质双分子层一致，区分了双分子层的碳氢化合物核心和界面区域。(4)阐明了(1)短TM螺旋（-12个氨基酸）和(2)来自KvAP电压门控K+通道的S4螺旋电压传感器肽的经位点辅助插入膜的物理基础。获得的信息将帮助我们了解如何预测不寻常的膜蛋白的拓扑结构，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.