STRUCTURE-BASED DESIGN OF PROTEIN FUNCTION

这项提议的重点是开发基于结构的设计和蛋白质功能工程的自动化设计技术。我们将通过在已知结构的蛋白质中引入新的配体结合位点，以及重新设计现有配体结合位点的性质来实现这一点。我们建议扩展我们之前的工作，其中我们已经成功地使用了基于结构的计算方法DEZYMER程序，以建立具有各种不同活性的金属中心在模型蛋白质的疏水核心。在这里，我们建议开发新的计算技术，将进一步重要的结构自由度引入到设计算法中。我们还建议开发基于结构的配体结合位点重新设计的新算法。这些程序将通过优化一系列合理设计的金属中心的稳定性和反应性，通过基于结构的几种不同蛋白质支架中的金属酶活性部位的设计，包括硫氧还蛋白、荧光绿色蛋白和脂肪酸结合蛋白，以及通过基于结构的细菌受体蛋白的特异性重新设计，来进行实验测试。这种方法的核心是设计周期，在设计周期中，建模和实验分析交替进行，从而迭代地测试和改进设计和计算方法。我们建议通过引入定向进化阶段来扩展这一方法，以通过实验选择和筛选方法来改进设计。基于结构的蛋白质结构和功能再设计方法的发展具有重要的工业和生物医学应用，如开发新型催化剂、生物传感器、新型生物材料和基于蛋白质的药物。

The focus of this proposal is to develop automated design techniques for the structure-based design and engineering of function in proteins. We will accomplish this by the introduction of new ligand-binding sites into proteins of known structure, and the redesign of the properties of existing ligand-binding sites. We propose to extend our previous work in which we have successfully used a structure-based computational approach, the DEZYMER program, to build metal centers with a variety of different activities in the hydrophobic core of a model protein. Here we propose to develop novel computational techniques that introduce further important structural degrees of freedom into the design algorithm. We also propose to develop novel algorithms for the structure-based redesign of ligand binding sites. This procedures will be tested experimentally by optimizing the stabilities and reactivities of a series of rationally designed metal centers; by structure-based design of metalloenzyme active sites in several different protein scaffolds, including thioredoxin, fluorescent green protein and fatty acid binding protein; and by the structure-based redesign of the specificity of a bacterial receptor protein. Central to this approach is the design cycle in which modeling and experimental analysis alternate, thereby iteratively testing and improving both the designs and the computational methods. We propose to extend this approach by introducing a directed evolution stage to improve the designs by experimental selection and screening methods. The development of methodologies for structure-based redesign of protein structure and function have important industrial and biomedical applications such as the development of new catalysts, biosensors, novel biomaterials, and protein-based pharmaceuticals.