EXPANDING THE CHEMISTRY OF NON-ENZYMATIC POLYUBIQUITINATION OF EXPRESSED PROTEINS FOR BIOCHEMICAL STUDIES.

The advances in chemical and semisynthetic approach of protein synthesis offer unique opportunities to deliver the desired bio-conjugates in high homogeneity and workable quantities to support biochemical and structural studies. These tools become more critical in systems where biological methods are unable to afford the target in high quality and quantities, as is the case with post-transnationally modified proteins such as ubiquitination. To address this formidable challenge we aim to develop and utilize highly efficient new chemical approaches to covalently attach ubiquitin chains to expressed protein substrates. Recently our group reported few methods to covalently link a protein substrate to polyubiquitine chains, but these approaches mostly limited to short ubiquitin chains or to a bio-chemically unstable conjugation linkage (e.g. disulphide).

To overcome these limitations we are currently exploring new approaches, which are based on selective modification of Cys residue of the protein of interest with an aldehyde moiety to enables oxime conjugation with a desired ubiquitin chain bearing hydroxylamine functionality at the C-terminus of ubiquitin. We applied this strategy to efficiently link tri- or tetra- ubiquitin chains to expressed α- and β-globin. Currently, we are using these bio-conjugates to understand the molecular basis of proeasomal degradation pathway.