Designing Affinity and Specificity at Protein Interfaces

描述(申请人提供)：蛋白质-蛋白质相互作用对几乎所有的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为研究细胞生物学和创造新的蛋白质疗法创造新的工具。这项研究的目标是开发和测试蛋白质界面设计的计算机协议，包括完整的主干和侧链灵活性。我们将在接口设计中的两个重要问题上测试这些协议：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，该程序用于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，该协议以前曾被用来设计一种具有原子级精度的新蛋白质结构。一些证据表明，当在模拟过程中考虑更多的侧链构象时，蛋白质设计模拟的准确性会提高。为了使侧链扭转角具有完全的灵活性，我们将使用蒙特卡罗最小化程序，在该过程中，离散的旋转体替换之后是基于梯度的侧链扭转角最小化。我们将通过设计两组模型蛋白质之间的功能正交结合作用来测试柔性侧链模型：泛素化途径的蛋白质和参与G蛋白信号转导的蛋白质。我们将通过设计蛋白质延伸来测试我们的蛋白质设计模型，该延伸通过增加与伴侣分子的有利接触数量来增强结合亲和力。具体地说，我们将在泛素结合酶UbcH7的N末端加入额外的残基，预计这些残基将增强其与其天然结合伙伴泛素连接酶E6AP的结合亲和力。结合亲和力将用各种生物物理技术来确定，活性分析将被用来确定重新设计的蛋白质是否作为活性复合体结合。

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.