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

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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.