A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that probably catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. Thus, understanding how inteins catalyze the steps in protein splicing may serve as a paradigm for understanding autoproteolysis mechanisms generally. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. We will address this situation by preparing semi-synthetic intein molecules containing unnatural amino acids and stable isotopic probes. Key to this part of the research program is the protein semisynthesis technique, expressed protein ligation, which allows unnatural amino acids and isotopic probes to be site-specifically introduced into large proteins. The availability of these segmental labeled molecules will allow us to conduct a series of isotope-edited NMR spectroscopy studies that are expected to reveal how inteins catalyze and regulate the protein splicing process both in cis and in trans. This information will not only deepen our understanding of protein splicing and related processes, but will also be useful for the further development of practical applications of protein splicing. Accordingly, in the last part of the program we will optimize and extend our recently introduced approach, conditional protein trans-splicing (CPS), that allows protein splicing to be triggered by the small molecule, rapamycin. Specifically, we will investigate whether CPS can be used to control protein function by developing a general on-switch for protein kinases. We will then explore whether the methodology works in living cells and what the scope and limitations are in this context. We anticipate that these studies will lead to the development of a general vehicle for controlling protein structure and function in vivo and will have numerous biological applications. [unreadable] [unreadable]