Computational design of beta-barrel membrane proteins

描述(申请人提供)：我们对膜蛋白所起的不同生物学作用的了解远远落后于我们对可溶性蛋白的了解，后者更易于表达、纯化和结晶。这里提出的研究是系统地探索表面氨基酸残基的疏水性在多大程度上决定了2桶蛋白质是保持可溶还是插入膜中。我们将对已知的2桶膜蛋白结构的表面氨基酸进行统计分析，从中我们将开发一种计算方法来评估将2桶蛋白插入膜中的能量成本。我们将应用这种方法重新设计大肠杆菌外膜蛋白OmpA的表面，使其具有不同的膜插入倾向。表达和评估每一种设计的膜结合将为优化我们的计算能量函数提供数据。使用我们经过验证的系统，我们将进行计算重新设计可溶的2桶蛋白GFP成为跨膜蛋白。将对设计的膜插入和荧光进行实验测试。通过荧光激活细胞分选的定向进化和高通量筛选将被用于逆转跨膜GFP中的任何荧光损失。这一研究的成功将代表着蛋白质设计和工程的重大成就，并为膜相关过程和膜蛋白质折叠的研究提供了强有力的工具。 与公共健康相关：膜蛋白是最常见的药物靶点之一，但我们对其特性的了解有限。我们的计算和实验研究将为膜蛋白的定义特征提供见解，并为膜相关过程的研究提供新的工具。

DESCRIPTION (provided by applicant): Our understanding of the diverse biological roles played by membrane proteins lags far behind our knowledge of soluble proteins, which are simpler to express, purify, and crystallize. The study proposed here is a systematic exploration of the extent to which the hydrophobicity of the surface amino acid residues determines whether a 2-barrel protein will remain soluble or insert into a membrane. We will perform a statistical analysis of the surface amino acids of known 2-barrel membrane protein structures, from which we will develop a computational method to evaluate the energetic cost of inserting a 2-barrel protein into a membrane. We will apply this method to redesign the surface of the E. coli outer membrane protein OmpA with "skins" of different membrane-insertion propensities. Expression and evaluation of the membrane incorporation of each of these designs will provide data to optimize our computational energy function. Using our validated system, we will undertake the computational redesign of the soluble 2-barrel protein GFP into a transmembrane protein. Designs will be experimentally tested for membrane insertion and fluorescence. Directed evolution and high-throughput screening by fluorescence-activated cell sorting will be used to reverse any loss of fluorescence in the transmembrane GFP. Success will represent a major achievement in protein design and engineering and provide a powerful tool for the study of membrane-associated processes and membrane protein folding. PUBLIC HEALTH RELEVANCE: Membrane proteins are among the most common drug targets, yet our understanding of their properties is limited. Our computational and experimental studies will provide insights on the defining characteristics of membrane proteins and provide new tools for the study of membrane-associated processes.