Mechanistic Studies and Directed Protein Evolution of a Semi-synthetic Intein

半合成内含肽的机理研究和定向蛋白质进化

• 批准号: 116694402
• 负责人: Professor Dr. Henning D. Mootz
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/116694402?language=en
• 关键词: Mechanistic; Studies; Directed; Protein; Evolution

Intein-catalyzed protein splicing follows a complex pathway with several intermediary steps, important aspects of which are only poorly understood. Inteins are widely used in diverse applications in the fields of protein purification, protein modification, and protein functional control. We will study the intein-catalyzed rearrangement of a peptide bond into a thio- or oxoester. This reaction represents the first and arguably most intriguing step of the protein splicing pathway. We will also focus on the coordination of the individual reaction steps, for which obviously several solutions exist. We will measure the reaction rates and determine the molecular groundwork for their functional coupling on a biochemical and a structural level. As a model system, a split intein will be used that is also useful for the semisynthesis of proteins. Its N-terminal fragment can be obtained by solid phase peptide synthesis. This allows the introduction of subtle structural changes in the active site through the incorporation of unnatural building blocks. An intein mutant with significantly improved catalytic properties was generated earlier in this project and will be included in the studies just like another novel split intein. Furthermore, new intein mutants will be selected by directed protein evolution to assess the impact of the mutations on the splicing mechanism. The progress in this project will also advance the potential of inteins in biotechnological applications.

内含肽催化的蛋白质剪接遵循一条包含多个中间步骤的复杂途径，但人们对其中的重要方面知之甚少。内含子广泛应用于蛋白质纯化、蛋白质修饰和蛋白质功能控制等领域。我们将研究内含肽催化的肽键重排为硫代酯或氧代酯。该反应代表了蛋白质剪接途径的第一步，也可以说是最有趣的一步。我们还将重点关注各个反应步骤的协调，显然存在多种解决方案。我们将测量反应速率并确定其在生化和结构水平上功能耦合的分子基础。作为模型系统，将使用分裂内含肽，它也可用于蛋白质的半合成。其N端片段可以通过固相肽合成获得。这允许通过结合非自然的构建块在活性位点引入微妙的结构变化。在该项目的早期，产生了具有显着改善的催化特性的内含肽突变体，并将像另一个新型分裂内含肽一样被纳入研究中。此外，将通过定向蛋白质进化选择新的内含肽突变体，以评估突变对剪接机制的影响。该项目的进展还将提升内含肽在生物技术应用中的潜力。