Poster abstracts

Poster number 31 submitted by Sourav Kumar Dey

A general method for site-specific labeling and conjugation of enzymes - exemplified by the lariat debranching enzyme Dbr1p

Sourav K. Dey (Department of Chemistry, Carnegie Mellon University), Debasish Grahacharya (Department of Chemistry, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University)

Abstract:
Enzymes that are site-specifically labeled using fluorescent dyes or biotin are particularly useful for bulk and single molecule biochemical investigations. However, the methods for site-specific labeling of enzymes use complex molecular biology techniques which can often result in enzymes with significant loss of function. Here we describe a straightforward and mild technique for N-terminal labeling and conjugation of a protein using a combination of native chemical ligation (NCL) and copper-mediated azide-alkyne cycloadditon ('click chemistry'). For the NCL reaction an azido lysine thioester molecule (azido-NCL-adapter) was synthesized starting from commercial Boc-Lysine in three simple steps. Initially, the condition for NCL using the azido-NCL-adapter was optimized with a test peptide that included an N-terminal cysteine. Subsequently, the peptide could be used in click-chemistry reactions that furnished labeled or conjugated peptide. With these optimized conditions we advanced to the lariat debranching enzyme (Dbr1p) that was expressed with an N-terminal cysteine. The Dbr1p cysteine reacted under NCL conditions with the azido-NCL-adapter to generate an azido-modified Dbr1p. This azido labeled enzyme could be conjugated either to a fluorescent dye (Alexa555) or to a biotinylated DNA sequence using 'click chemistry'. The biotin-DNA tether on the N-terminus of Dbr1p can serve to immobilize the enzyme on a surface for single molecule experiments. The Dbr1p specifically cleaves the 2'-5' phosphodiester bond of lariat RNA. The modified enzyme - labeled or conjugated - showed retention of the debranching activity in cleaving 2'-5' phosphodiester linkage of a backbone branched RNA substrate. Such site-specifically modified debranching enzymes will be used for single molecule experiments in the future. As well, we envision our straightforward method for site-specific labeling or conjugation will find more general use for the investigation of other proteins.

Keywords: Native chemical ligation, click-chemistry, site specific labeling