PI: Dr. Champak Chatterjee
Location: UW Chemistry
Website: http://blogs.uw.edu/champak1/

Chemoenzymatic Strategies for Site-selective Protein Modification and Bioconjugation

The ability to modify proteins in a site- and chemistry-specific manner permits the introduction of diverse probes of protein function/structure and for protein conjugation with various biologically active molecules. The recent discovery of the prokaryotic ubiquitin-like protein (Pup) modifier in Actinomycete bacteria (1) has led to the identification of a novel class of protein ligase and protease enzymes that have no eukaryotic homologs and are potential tools for reversible and site-specific protein modification. The specific project goals are to utilize our knowledge of the interaction between the ligase and Pup (2) to produce chimeric proteins that are stoichiometrically and site-specifically modified by various small-molecules. Examples include antibody-drug conjugates and fluorophore- or spin probe-labeled cell-surface receptors. In another line of inquiry, we will elucidate the molecular mechanism underlying target lysine selection by the Pup ligase in its known substrates using the tools of mutagenesis, NMR, and X-ray crystallography. By understanding the rules governing specificity for a single lysine side-chain in substrates containing multiple solvent-exposed lysines, we will achieve predictability and control in protein labeling by Pup and its chimeras. The long-term goal would then be to harness our mechanistic knowledge toward re-engineering the specificity of the Pup ligase for target lysines in novel contexts and thereby generate a suite of engineered ligases for labeling proteins at precisely defined sites.

(1) Pearce, M. J.; Mintseris, J.; Ferreyra, J.; Gygi, S. P.; Darwin, K. H. (2008) Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis, Science 322, 1104.
(2) Smirnov, D.; Dhall, A.; Sivanesam, K.; Sharar, R.J.; Chatterjee, C. (2013) Fluorescent probes reveal a minimal ligation recognition motif in the prokaryotic ubiquitin-like protein from Mycobacterium tuberculosis. J. Am. Chem. Soc. 135, 2887-2890.