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

内含素催化的蛋白质剪接遵循一条复杂的途径，包括几个中间步骤，其中重要的方面还知之甚少。内含子在蛋白质纯化、蛋白质修饰、蛋白质功能调控等领域有着广泛的应用。我们将研究内含素催化的多肽键重排为硫代酯或氧化酯。这个反应代表了蛋白质剪接途径的第一步，也是最耐人寻味的一步。我们还将重点讨论各个反应步骤的协调，显然存在几种解决方案。我们将测量反应速度，并在生化和结构水平上确定它们功能偶联的分子基础。作为一个模型系统，将使用对蛋白质半合成也有用的分裂内含子。其N-末端片段可通过固相多肽合成得到。这允许通过加入非自然的构建块在活动站点中引入微妙的结构变化。在这个项目的早期已经产生了一个催化性能显著改善的内含素突变体，并将像另一个新的分裂内含子一样被包括在研究中。此外，将通过定向蛋白质进化来选择新的内含素突变体，以评估突变对剪接机制的影响。该项目的进展还将促进整合素在生物技术应用中的潜力。

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.