Designing Affinity and Specificity at Protein Interfaces

描述（由申请人提供）：蛋白质-蛋白质相互作用对几乎所有细胞过程都是必不可少的。合理操纵这些相互作用的能力将允许创建用于研究细胞生物学的新工具和创建新的蛋白质疗法。本研究的目的是开发和测试计算机协议的蛋白质接口设计，包括完整的骨干和侧链的灵活性。我们将测试这些协议在接口设计中的两个重要问题：增强自然发生的相互作用的结合亲和力和改变信号蛋白的结合特异性。为了实现这一目标，我们将利用我们开发的计算机程序Rosetta Design来同时优化目标结构的氨基酸序列和骨架构象。令人鼓舞的是，该协议以前已被用于设计具有原子级精度的新型蛋白质结构。一些证据表明，蛋白质设计模拟的准确性增加时，更多的侧链构象被认为是在模拟过程中。为了允许侧链扭转角的完全灵活性，我们将使用Monte Carlo最小化程序，其中离散旋转异构体取代之后是侧链扭转角的基于梯度的最小化。我们将通过设计两组模型蛋白质之间的功能正交结合相互作用来测试柔性侧链模型：来自泛素化途径的蛋白质和参与G蛋白信号传导的蛋白质。我们将通过设计蛋白质延伸来测试我们的具有柔性骨架的蛋白质设计模型，所述蛋白质延伸通过增加与伴侣分子的有利接触的数量来增强结合亲和力。具体地说，我们将纳入额外的残基关闭的N-末端的泛素缀合酶，UbcH 7，这是预测，以提高其结合亲和力，其天然的结合伴侣，泛素连接酶E6 AP。结合亲和力将用各种生物物理技术测定，并且活性测定将用于测定重新设计的蛋白质是否作为活性复合物结合。

DESCRIPTION (provided by applicant): Protein-protein interactions are essential to almost all cellular processes. The ability to rationally manipulate these interactions would allow for the creation of new tools for studying cell biology and the creation of new protein therapeutics. The objective of this research is to develop and test computer protocols for protein interface design that include full backbone and side chain flexibility. We will test these protocols on two important problems in interface design: enhancing the binding affinity of naturally occurring interactions and changing the binding specificity of signaling proteins. To achieve this objective we will make use of a computer program, Rosetta Design, that we developed for simultaneously optimizing the amino acid sequence and backbone conformation of a target structure. Encouragingly, this protocol has been previously used to design a novel protein structure with atomic level accuracy. Several lines of evidence suggest that the accuracy of protein design simulations increase when more side chain conformations are considered during the simulation. To allow complete flexibility in side chain torsion angles we will use a Monte Carlo minimization procedure in which discrete rotamer substitutions are followed by gradient-based minimization of side chain torsion angles. We will test the flexible side chain model by designing functionally orthogonal binding interactions between two sets of model proteins: proteins from the ubiquitination pathway and proteins involved in G-protein signaling. We will test our model for protein design with a flexible backbone by designing protein extensions that enhance binding affinity by increasing the number of favorable contacts with the partner molecule. Specifically, we will incorporate additional residues off the N-terminus of the ubiquitin conjugating enzyme, UbcH7, that are predicted to enhance its binding affinity for its natural binding partner, the ubiquitin ligase E6AP. Binding affinities will be determined with various biophysical techniques and activity assays will be used to determine if the redesigned proteins bind as an active complex.