Structural Requirements for Protein Membrane Assembly

本研究的重点是细菌膜蛋白的n端尾部如何在膜双分子层上转移。目前尚不清楚这些尾部的膜插入是否需要SecA或SecY （sec机制的中心成分），或者插入是否可以自发地发生在脂质双分子层中。大多数具有n端周质尾部的蛋白质符合“内正”规则，与周质环相比，胞质环上的带正电残基更多。然而，球形Rhodobacter sphaeroides光合反应中心的H亚基违反了这一规则，它含有一个n端周质尾，在膜/细胞质边界有一个高负电荷区域。本提案的目的是：1)确定短n端尾部是通过膜内的脂质插入还是通过蛋白质复合物插入；2)定义n端尾部的特征（长度、电荷或两亲性螺旋），这些特征是尾部在膜上转移所需要的；3)检查膜蛋白是否存在一定长度的n端尾部，在那里膜蛋白利用sec机制；4)确定H亚基的蛋白质拓扑结构的决定因素，H亚基是一种取向不符合“内部正”规则的蛋白质。将使用多种方法，包括定点诱变、基因融合、拓扑作图和无蛋白脂质体的生化研究。这个项目的重点是细菌膜蛋白是如何插入到膜上的。生物膜的精细结构，以及膜蛋白在生物膜上的精确定位，对生物系统的正常运作至关重要。理解大自然用来实现这种定义架构的“规则”，对于成功利用现代基因工程和生物处理技术至关重要。该研究项目将有助于更好地理解膜蛋白如何实现其最终拓扑结构和位置的自然规则。＊＊＊

9316891 Dalbey The proposed research is focused on how N-terminal tails of bacterial membrane proteins are transferred across the membrane bilayer. It is not known whether membrane insertion of these tails will require SecA or SecY, central components of the sec- machinery, or whether insertion can occur spontaneously into the lipid bilayer. Most proteins with N-terminal periplasmic tails conform to the "positive inside" rule with mroe positively charged residues on cytosolic loops compared to periplasmic loops. However, subunit H of the photosynthetic reaction center of Rhodobacter sphaeroides, which contains an N-terminal periplasmic tail, violates this rule and has a highly negatively charged region at the membrane/cytoplasm border. The aims of this proposal are: 1), to determine whether short N-terminal tails are inserted through the lipid or through a proteinaceous complex within the membrane; 2), to define the features (length, charge, or an amphiphilic helix) within N-terminal tails that are needed to translocate the tails across the membrane; 3), to examine whether there is a certain length of the N-terminal tails where the membrane proteins utilize the sec- machinery; and 4), to identify the determinants of the protein topology of subunit H, a protein that has an orientation that disobeys the "positive inside" rule. A variety of approaches will be used, including site-directed mutagenesis, gene fusions, topological mapping, and biochemical studies with protein- free liposomes. %%% This project focuses on the question of how bacterial membrane proteins are inserted into membranes. The delicate architecture of biological membranes, and the fine- resolution accuracy of placement of membrane proteins in biological membranes, are absolutely critical to the proper functioning of biological systems. An understanding of the "rules" that Nature uses to achieve this defined architecture is in turn critical to the successful exploitation of modern gen etic engineering and bioprocessing technologies. This research projects will result in better understanding those rules of Nature concerning how membrane proteins achieve their final topology and location. ***