In Vitro Selection of Proteins via mRNA-Protein Fusions

分子设计中最具挑战性的问题之一是开发出 可以结合蛋白质表面并阻止蛋白质之间的相互作用。调节的能力 蛋白质-蛋白质相互作用以定向的方式开启了探测和 通过设计控制生物系统。这项提议的长期目标是 是使用体外选择实验来靶向蛋白质表面，探索基本的 蛋白质识别、结构、功能、特异性和催化等方面。离体 遗传方法目前代表了一种强大的蛋白质操作解决方案 设计问题。此前，PI已经构思、开发和实施了mRNA- 多肽与蛋白质融合(以下简称“信使核糖核酸展示”)体外设计蛋白质 选拔实验。与其他生物相比，信使核糖核酸显示具有重要的优势 体外和体内蛋白质设计策略，如检测能力非常大 文库(&gt；1013个单独的序列)，在没有活细胞的情况下，具有紧密的 对捆绑和严格性的实验控制。在这项提案中，我们将继续 将G蛋白连锁信号作为配基设计的靶点。我们的目标是探索 蛋白质识别的生物物理化学，因为它与这一重要信号有关 路径。我们的具体目标是： 1)增强我们的G蛋白和GPCR导向的配体的性质。 2)开发针对2-肾上腺素能的多肽和蛋白质配体 (2)G蛋白偶联受体(GPCR)。 3)研究GPCR导向的配体的功能和结构。

One of the most challenging problems in molecular design is developing molecules that can bind protein surfaces and block protein-protein interactions. The ability to modulate protein-protein interactions in a directed fashion opens the possibility of probing and controlling biological systems through design. The long- term objective of this proposal is to use in vitro selection experiments to target protein surfaces, exploring fundamental aspects of protein recognition, structure, function, specificity, and catalysis. In vitro genetic approaches currently represent a powerful operational solution to the protein design problem. Previously, the PI has conceived, developed, and implemented mRNA- peptide and protein fusions (hereafter "mRNA display) to design proteins using in vitro selection experiments. mRNA display provides important advantages relative to other in vitro and in vivo protein design strategies, such as the ability to examine very large libraries (>1013 individual sequences) in the absence of a living cell, with tight experimental control over binding and stringency. In this proposal, we will continue using G-protein linked signaling as a target for ligand design. Our goal is to explore the biophysical chemistry of protein recognition as it pertains to this important signaling pathway. Our specific aims are: 1) To enhance the properties of our G protein and GPCR-directed ligands. 2) To develop peptide and protein ligands targeting the 2-adrenergic (2AR) G protein coupled receptor (GPCR). 3) To explore the function and structure of our GPCR-directed ligands.