INCORPORATION OF NONNATURAL AMINO ACIDS INTO PROTEINS

将非天然氨基酸掺入蛋白质中

• 批准号: 2684918
• 负责人: A RICHARD CHAMBERLIN
• 金额: $ 15.79万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684918
• 关键词: FK506; aminoacid analog; calcineurin; cytochrome c; cytochrome c peroxidase; fluorescent dye /probe; peptide chemical synthesis; peptidylprolyl isomerase; photochemistry; point mutation; protein engineering; protein sequence; suppressor mutations; synthetic peptide

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).

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 基础机械酶学和商业上结构修饰 基因工程 尽管这项技术意义重大， 受到氨基酸取代限于 20种主要氨基酸 这一先决条件通常排除了 将“设计者”氨基酸直接定点导入蛋白质 意图以可预测的方式改变功能或活动。 这 提案要求提供资金，以继续我们的工作， 将非天然氨基酸掺入蛋白质的技术。 在 在过去的四年里，我们评估了使用半 合成抑制子与体外翻译系统联合用于 这个目的。 我们还开始了几个合作项目， 这项技术被用于制备突变蛋白质， 三种不同蛋白质的结构/功能研究。 期间 我们将对现有的资金进行一些微调， 程序，包括努力i）提高抑制剂的效率 合成和ii）开发用于增加蛋白质生产的一般方法 和/或抑制效率。 主要重点将是应用 技术：iii）研究蛋白质-蛋白质结合之间的相互作用， 通过光化学交联的FK 506-FKBP复合物和钙调磷酸酶，iv） 细胞色素C到细胞色素C过氧化物酶电子传递研究 (CCP)假设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 单个位点的固有荧光探针，最初应用于 制备可溶性组织因子（sTF）的突变体。