Structure-based design of protein function

描述（由申请人提供）：在我的实验室开发的基于结构的计算蛋白质设计方法的最新进展，成功地预测了急剧改变受体蛋白的配体结合特异性的突变。利用这些设计技术，E.通常结合糖或氨基酸的大肠杆菌周质结合蛋白（PBP）超家族已被转化为以高亲和力和特异性识别化学上不同的配体的受体。荧光和电化学报告基团的引入允许工程PBPs被用作无试剂光学或生物电子生物传感器。这些受体也可以重新导入E.在大肠杆菌中，它们控制介导对非天然的细胞外化学信号的转录激活反应的合成信号转导途径。这些早期的结果是非常令人鼓舞的，并表明，各种各样的生物功能的计算设计可以考虑。然而，为了使这种能力成为现实，有必要进一步发展计算设计技术，并将其扩展到处理越来越复杂的结合位点，如蛋白质-蛋白质和蛋白质-DNA相互作用。以高精度和复杂度设计蛋白质的能力具有许多生物医学应用。我建议进一步开发和实验测试用于操纵蛋白质分子识别的计算设计技术，使用特定的，生物医学相关的应用程序作为设计目标，指导将被工程化的受体系统和配体的选择。因此，下面确定的任务旨在具有明确的实际应用，说明计算蛋白质工程对生物医学科学的潜在广泛效用，同时探索有关蛋白质分子识别的基本科学问题。目的1-3集中于开发具有显著改变的配体结合特性的受体。这些探索了临床科学（目标1）、药理学（目标2）和细胞生物学（目标3）中的不同应用。从基础科学的角度来看，它们将使我们能够开发工程结合位点的技术，并探索PBPs以外的支架（目标2）。目标4旨在将设计技术扩展到更复杂的系统。

DESCRIPTION (provided by applicant): Recent advances in computational, structure-based protein design methods, developed in my laboratory, successfully predict mutations that drastically alter the ligand-binding specificity of receptor proteins. Using these design techniques, several members of the E. coli periplasmic binding protein (PBP) superfamily that normally bind sugars or amino acids have been converted into receptors that recognize chemically diverse ligands with high affinity and specificity. Introduction of fluorescent and electrochemical reporter groups has permitted the engineered PBPs to be used as reagentless optical or bioelectronic biosensors. The receptors also can be re-introduced into E. coli where they control synthetic signal transduction pathways that mediate transcriptional activation response to non-natural, extracellular chemical signals. These early results are highly encouraging and suggest that the computational design of a wide variety of biological functions can be contemplated. However, in order for this capability to become a reality, it is necessary to further develop the computational design techniques, and to extend them to dealing with binding sites of increasing complexity, such as protein-protein and protein-DNA interactions. The ability to engineer proteins with a high degree of precision and sophistication has numerous biomedical applications. I propose to further develop and experimentally test the computational design techniques for manipulating molecular recognition in proteins, using specific, biomedically relevant applications as design targets that guide the choice of receptor systems and ligands that will be engineered. The tasks identified below are therefore intended to have clear practical applications, illustrating the potential wide-ranging utility of computational protein engineering to the biomedical sciences, while at the same time exploring basic scientific questions regarding molecular recognition in proteins. Aims 1-3 are focused on the development of receptors with drastically altered ligand-binding properties. These explore different applications in clinical science (Aim 1), pharmacology (Aim 2), and cell biology (Aim 3). From a basic science point of view, they will allow us to develop the techniques for engineering binding sites, and to explore scaffolds other than the PBPs (Aim 2). Aim 4 is intended to extend the design technique to much more complex systems.