CAA: Synthetic Signal Peptides: Probes of Molecular Interactions During Secretion

小行星9629740 这是一个为女性科学家和工程师设立的职业发展奖。 该项目关注蛋白质如何被靶向并跨细胞膜转运。 在大多数情况下，无论所讨论的膜是细菌膜，真核内质网，或线粒体或叶绿体膜，这种蛋白质转运系统的统一特征是氨基末端信号肽参与蛋白质的运输。 在跨真核内质网转运的情况下，信号肽在转运开始之前与信号识别颗粒（SRP）相互作用。 拟议的研究旨在阐明细菌分泌蛋白的信号肽和P48（也称为Ffh），哺乳动物信号识别颗粒（SRP）的54 kD蛋白质的土方ischia coli同源物之间的相互作用。 越来越多的证据表明，P48在蛋白质分泌的初始阶段起着至关重要的作用，通过识别输出蛋白的信号肽区域。 将使用一系列模型合成信号肽研究这种相互作用，所述信号肽在其疏水性程度上系统地变化，并且代表体内不同水平的分泌能力。 这些肽将进行化学合成、纯化和表征，以验证其物理性质，包括二级结构。 将基于放射性标记肽的置换开发结合试验。 Scatchard分析将用于确定信号肽-P48对在不同条件下的解离常数，包括4.5S RNA的缔合和各种形式的鸟嘌呤核苷酸的存在。 随后，将使用化学交联方法来定位P48上参与信号肽相互作用的位置。 这些研究将有助于我们了解大肠杆菌蛋白质分泌的早期事件。大肠杆菌中，并在几个其他系统中，已发现有一个SRP同源。 ***

9629740 Kendall This is a Career Advancement Award for Women Scientists and Engineers. This project concerns how proteins are targeted to and transported across cell membranes. In most cases, whether the membrane in question is the bacterial membrane, the eukaryotic endoplasmic reticulum, or the mitochondrial or chloroplast membrane, a unifying feature of such protein transport systems is the involvement of an amino-terminus signal peptide on the protein to be transported. In the case of transport across the eukaryotic endoplasmic reticulum, the signal peptide interacts with a signal recognition particle (SRP) prior to the initiation of transport. The proposed studies are aimed at elucidating the interaction between the signal peptide of a bacterial secreted protein and P48 (also called Ffh), the Escherischia coli homologue of the 54 kD protein of the mammalian signal recognition particle (SRP). Like its mammalian counterpart, there is growing evidence that P48 plays a vital role in the initial stages of protein secretion through recognition of the signal peptide region of export-competent proteins. This interaction will be investigated using a series of model synthetic signal peptides which vary systematically in their degree of hydrophobicity and which represent different levels of secretion competence in vivo. These peptides will be chemically synthesized, purified, and characterized to verify their physical properties, including secondary structure. A binding assay will be developed based on displacement of a radiolabeled peptide. A Scatchard analysis will be used to determine the dissociation constant for the signal peptide-P48 pair under different conditions, including the association of 4.5S RNA and the present of various forms of guanine nucleotides. Subsequently,, a chemical crosslinking approach will be used to map the location on P48 involved in the signal peptide interaction. These studies will help in our understanding of early events in protein secretion in E. coli and in the several other systems which have been found to have an SRP homologue. ***