Inteins: Expanding Biological Roles and Biotechnological Applications - PROJECT SUMMARY/ABSTACT The long-term goals of this application are to better understand the biological importance of inteins as post- translational regulatory elements, their emerging role in pathogen stress response, and to harness the power of their unique chemistry to invent useful technologies for the biomedical research community. Inteins, or intervening proteins, are self-catalytic, mobile genetic elements removed from host genes through protein splicing. From the applied perspective, the ability of inteins to shuffle peptide bonds in highly specific ways has proven exceptionally useful in protein engineering, leading to the development of numerous technologies. While intein applications have dominated their investigation, and new intein-based technologies are developed frequently, the biological importance of inteins in nature is poorly understood. Inteins are abundant mobile genetic elements in the microbial world, found in approximately one-half of archaea and one-quarter of bacteria. Contrary to long-standing assumption that inteins are molecular parasites, mounting recent evidence suggests that some intein-containing proteins have evolved to couple splicing, and thus host protein activation, to environmental signals. This represents a novel and potentially widespread form of post-translational regulation. Further, as inteins are absent in humans and located within essential genes of several pathogens, understanding environmental factors that regulate protein splicing may inform antimicrobial development. We propose the following three aims, building upon recent discoveries, as well as expanding into new arenas. Aim 1 challenges the assumption that unspliced precursor proteins are inert, for the first time systematically investigating the potential functions of an intein-containing DNA helicase prior to splicing. Aim 1 will also examine the importance of protein splicing within the natural context of the intein, determining the consequences of intein deletion and mutation on host physiology. Aim 2 will systematically examine splicing of all fifteen inteins from a model archaeon to examine the diversity of splicing within one host, as well as examine potential regulation. Aim 3 seeks to expand upon a recently developed intein-based protein stability biosensor. Through these Aims, this work will enhance our understanding of the roles these exciting and understudied elements play in nature, as well as develop a new intein-based biosensor to examine and improve protein folding in vivo.
