INCORPORATION OF NONNATURAL AMINO ACIDS INTO PROTEINS

定点突变是最重要的实验工具之一。 可用于蛋白质研究，作为选择性的基石 基础机械酶学和商品化的结构修饰 基因工程。尽管它具有巨大的意义，但这种技术 受制于氨基酸替换仅限于 二十种主要氨基酸。此前提条件通常不包括 直接定点导入“设计型”氨基酸的蛋白质 意在以可预测的方式修改功能或活动。这 提案要求提供资金，以继续我们的开发和应用工作 蛋白质中加入非天然氨基酸的技术。在……里面 在过去的四年里，我们已经评估了使用Semi-Of的一般性 合成抑制子与体外翻译系统相结合 这就是目的。我们还开始了几个合作项目，其中 这项技术正被用来制备突变蛋白质 三种不同蛋白质的结构/功能研究。在此期间 对于所要求的资金，我们将对目前的 程序，包括努力：一)提高抑制者的效率 合成和二)开发提高蛋白质产量的一般方法 和/或抑制效率。主要的重点将是应用 技术：iii)研究蛋白质-蛋白质结合相互作用 FK506-FKBP络合物和钙调神经磷酸酶光化学交联，iv) 细胞色素C与细胞色素C过氧化物酶的电子传递研究 (CCP)提出的氢键途径被破坏的突变体 实际上，通过单原子突变，以及v)研究引入 在单个位置的本征荧光探针，最初应用于 制备可溶性组织因子(STF)突变体。

Site-specific mutagenesis is one of the most important experimental tools available for protein research, serving as the cornerstone for selective structural modification in both basic mechanistic enzymology and commercial genetic engineering. Despite its tremendous significance, this technique suffers from the limitation that amino acid substitutions are restricted to the twenty primary amino acids. This prerequisite normally excludes the direct site-specific introduction into proteins of "designer" amino acids intended to modify function or activity in a predictable way. This proposal requests funding to continue our work on developing and applying techniques for incorporation of nonnatural amino acids into proteins. In the previous four years we have assessed the generality of using semi- synthetic suppressors in conjunction with in vitro translation systems for this purpose. We have also begun several collaborative projects in which this technique is being used to prepare mutant proteins for structure/function studies of three different proteins. During the period of funding requested, we will carry out some fine-tuning of current procedures, including efforts to i) improve efficiency of suppressor synthesis and ii) develop general methods for increasing protein production and/or suppression efficiency. The primary focus will be on applying the technique to: iii) study the protein-protein binding interaction between FK506-FKBP complex and calcineurin via photochemical crosslinking, iv) investigate electron transfer from cytochrome C to cytochrome C peroxidase (CCP) mutants in which proposed hydrogen bond pathways have been disrupted by, in effect, single atom mutations, and v) investigate the introduction of intrinsic fluorescence probes at single sites, initially applied to preparing mutants of soluble tissue factor (sTF).