Computational design of beta-barrel membrane proteins

描述（由申请人提供）：我们对膜蛋白多种生物学作用的了解远远落后于我们对可溶性蛋白的了解，可溶性蛋白更容易表达、纯化和结晶。这里提出的研究是对表面氨基酸残基的疏水性在多大程度上决定2桶蛋白是否保持可溶性或插入膜的系统探索。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桶膜蛋白插入膜的能量成本。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。表达和评估这些设计的膜结合将为优化我们的计算能量函数提供数据。使用我们验证过的系统，我们将对可溶性2桶蛋白GFP进行计算重新设计，使其成为跨膜蛋白。设计将通过实验测试膜插入和荧光。定向进化和荧光激活细胞分选的高通量筛选将用于逆转跨膜GFP中荧光的任何损失。成功将代表蛋白质设计和工程的重大成就，并为膜相关过程和膜蛋白折叠的研究提供有力的工具。

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.