Department of Chemistry, Princeton University, Princeton, New Jersey 08544-1009, USA.
Protein Sci. 2011 Apr;20(4):702-11. doi: 10.1002/pro.601. Epub 2011 Mar 7.
A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis will require proteins that recognize and bind to small molecules. Here, we show that stably folded α-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this four-helix bundle. The parental protein and the Phe→Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.
蛋白质设计的一个核心目标是设计新型蛋白质，以应用于生物技术和医学。许多应用，包括那些专注于传感和催化的应用，都需要能够识别和结合小分子的蛋白质。在这里，我们表明，从设计序列的二进制图案文库中分离出的稳定折叠的α-螺旋蛋白质可以突变产生能够结合一系列小芳香族化合物的结合位点。具体来说，我们在从头设计的蛋白质 S-824 的已知结构中突变了两个苯丙氨酸侧链为丙氨酸，在这个四螺旋束的核心中产生了埋藏的空腔。原始蛋白和 Phe→Ala 变体暴露于化合物混合物中，并通过饱和转移差 NMR 评估选择性结合。通过脉冲场梯度自旋回波 NMR 测定了苯及其一些衍生物的亲和力，并且证明其中一些化合物与突变蛋白以微摩尔离解常数结合。这些研究表明，来自二进制图案文库的稳定折叠的从头设计蛋白质非常适合作为结合位点设计的支架。
