Structural Requirements for Protein Membrane Assembly

小行星9316891 拟议的研究重点是如何N-末端尾巴的细菌膜蛋白跨膜双层转移。 目前尚不清楚这些尾的膜插入是否需要SecA或SecY，SecA或SecY是sec机制的中心组分，或者插入是否可以自发地发生在脂质双层中。 大多数具有N-末端周质尾的蛋白质符合“正内部”规则，与周质环相比，胞质环上的残基更多带正电荷。 然而，亚基H的光合反应中心的球形红细菌，其中包含一个N-末端周质尾，违反了这一规则，并具有高度负电荷的区域在膜/细胞质边界。 该建议的目的是：1），确定短的N-末端尾是通过脂质还是通过膜内的蛋白质复合物插入; 2），确定膜内的特征。（长度、电荷或两亲性螺旋），所述两亲性螺旋在N末端尾内，所述N末端尾是使尾易位穿过膜所需的;（3）检查是否存在一定长度的N-末端尾，在那里膜蛋白利用sec-机制;和4）鉴定亚基H的蛋白质拓扑结构的决定因素，亚基H是具有不遵守“正内部”规则的取向的蛋白质。 将使用多种方法，包括定点诱变、基因融合、拓扑作图和使用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膜的问题。 生物膜的精细结构和膜蛋白在生物膜中的位置的精细分辨率准确性对于生物系统的正常功能是绝对关键的。 理解自然界用来实现这种定义的结构的“规则”反过来对成功利用现代遗传工程和生物加工技术至关重要。 该研究项目将导致更好地理解自然界关于膜蛋白如何实现其最终拓扑结构和位置的规则。 ***

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. ***