Chemically modified protein derivatives represent an important class of molecules with applications ranging from therapeutic development to basic biomedical research. Technologies for the construction of modified proteins are therefore highly desirable. The use of enzymes for installing non-natural protein modifications has gained traction in recent years due to the remarkable site-selectivity that enzymes afford. Among enzymes reported for this purpose, bacterial sortases have garnered significant attention because of their selectivity, and their ability to install a wide range of chemical modifications. While promising, this technology is subject to limitations stemming from the inherent reversibility of the chemistry, and the inability of commonly used sortases to efficiently modify sites other than the termini of protein targets. Circumventing these limitations is critical to the further refinement of sortase-based technology. Addressing these issues would also represent an important step in our long-term effort to exploit sortase reactivity for the construction of protei derivatives for a range of therapeutic, diagnostic, biomaterials, and basic research applications. Specifically, the objective of this proposal is to develop strategies for 1) controlling the equilibrium of sortase-catalyzed ligation reactions and for 2) targeting internal amino acid positions through the controlled formation of isopeptide bonds. We will achieve this objective through two specific aims. First, we will explore the incorporation of masked metal binding peptides into sortase substrate motifs. This will allow for control of reaction equilibrium through the deactivation of critical reaction by-products. These studies will include a rigorous optimization of this metal promoted strategy, an exploration of its compatibility with full size protein targets, and its application to the synthesis of unique polypeptides with potential for use as medical adhesives. Second, we will exploit the reactivity of naturally occurring sortase homologs as a means for efficiently generating isopeptide bonds with nucleophilic lysine residues. Using a combination of synthetic peptides and recombinant protein targets, we will characterize the ability of sortase homologs from Streptococcus suis and Streptococcus oralis to selectively generate isopeptide bonds in vitro. The innovative aspects of this proposal are the novel application of metal peptide complexes for blocking reaction reversibility, and the use of naturally occurring sortase homologs that have never been studied in the context of protein modification chemistry. The proposed research is also significant because it will substantially improve the efficiency and scope of sortase-based strategies. Overall, these studies will provide a foundation for powerful new protein modification methods, and will enhance our fundamental understanding of sortase reactivity and its implementation in protein modification chemistry.