A versatile approach to transform low-affinity peptides into protein probes with cotranslationally expressed chemical cross-linker

Abstract

The potential usefulness of artificially selected peptides as probes to detect specific proteins has been proposed because of the ease and low cost of syntheses, manipulation and genetic expression. However, the affinities of these peptides to their target proteins are generally too low to be practical as diagnostic or bio-analytical reagents. One approach to this problem is to incorporate a redox-active amino acid 3,4-dihydroxy-L-phenylalanine (L-DOPA) which selectively forms a covalent linkage to the target protein. Such peptide-based probes can also be fused to tailored reporter proteins and easily expressed in bacterial cultures. As a demonstration, a candidate peptide TOP1 that weakly binds to the target protein, the SH3 domain of human Abl kinase, was fused to green fluorescent protein (GFP) and L-DOPA was site-specifically incorporated into the peptide region (TOP1-DOPA-GFP). TOP1-DOPA-GFP produced from E. coli was used in a Western blot-type experiment to show that the Abl SH3 domain can be detected in one step by observing the fluorescence. The molecular design presented in this work is significant in that the same approach could be used to transform many other protein-binding peptides with insufficient affinities into protein detection probes with a variety of fused reporter or therapeutic proteins.

Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

“…Direct comparison of the crosslinking efficiencies obtained herein to previously published results proved difficult because crosslinking efficiencies have generally not been evaluated quantitatively, but rather just examined qualitatively by gel mobility shifts (Burdine et al, 2004; Umeda et al, 2009, 2010). Our results therefore likely represent the first quantitative study of l -DOPA crosslinking.…”

“…While there have been several successful demonstrations of the rational placement of crosslinking amino acids into proteins (Alfonta et al, 2003; Forne et al, 2012; Sato et al, 2011; Umeda et al, 2009, 2010), to date there has not been a thorough analysis of how a detailed structural analysis might be used to guide the placement of amino acids that would crosslink efficiently. Currently, placement of NCAAs into proteins is based on intuition and no computational tools exist to guide the design of crosslinks.…”

“…l -DOPA has previously been used to successfully crosslink the monomeric domains of a dimeric sortase A for structural studies (Umeda et al, 2009), to enhance the affinity of low-affinity peptide probes for a kinase SH3 bioassay (Umeda et al, 2010), and to site-specifically label proteins with polysaccharides (Ayyadurai et al, 2011). While previously reported uses of l -DOPA as a site-specific crosslinker have yielded examples of effective crosslinking (as shown by SDS–PAGE or Western blot analyses), the actual efficiencies of crosslinking have never been reported (Burdine et al, 2004; Umeda et al, 2009, 2010).…”

“…While previously reported uses of l -DOPA as a site-specific crosslinker have yielded examples of effective crosslinking (as shown by SDS–PAGE or Western blot analyses), the actual efficiencies of crosslinking have never been reported (Burdine et al, 2004; Umeda et al, 2009, 2010). These previous reports indicated that it was possible to place l -DOPA by intuition, but did not provide more quantitative assessments of what parameters impacted crosslinking efficiency (Umeda et al, 2009, 2010). …”