Smadbeck James, Peterson Meghan B, Khoury George A, Taylor Martin S, Floudas Christodoulos A
Department of Chemical and Biological Engineering, Princeton University, USA.
J Vis Exp. 2013 Jul 25(77):50476. doi: 10.3791/50476.
The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity. To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
从头蛋白质设计的目标是找到能够折叠成所需三维结构的氨基酸序列,相对于天然序列,其在特定性质上有所改进,如结合亲和力、激动剂或拮抗剂行为或稳定性。蛋白质设计是当前药物设计与发现进展的核心。蛋白质设计不仅为潜在有用的药物靶点提供预测,还增进了我们对蛋白质折叠过程和蛋白质 - 蛋白质相互作用的理解。诸如定向进化等实验方法在蛋白质设计中已取得成功。然而,此类方法受到可有效搜索的有限序列空间的限制。相比之下,计算设计策略允许筛选涵盖广泛性质和功能的更大序列集。我们已开发出一系列能够解决蛋白质设计几个重要领域问题的从头计算蛋白质设计方法。这些包括设计具有更高稳定性的单体蛋白质以及具有更高结合亲和力的复合物。为了更广泛地传播这些方法以供使用,我们推出了Protein WISDOM(http://www.proteinwisdom.org),这是一个为各种蛋白质设计问题提供自动化方法的工具。提交结构模板以初始化设计过程。设计的第一阶段是优化序列选择阶段,其目的是通过最小化序列空间中的势能来提高稳定性。然后将选定的序列经过折叠特异性阶段和结合亲和力阶段。该过程每个步骤的序列排名列表以及相关的设计结构为用户提供了对设计的全面定量评估。在此我们提供每种设计方法的详细信息,以及通过使用这些方法取得的几个显著实验成功案例。