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 bioanalytical reagents. One approach to this problem is to incorporate a redox-active amino acid, 3,4-dihydroxy-l-phenylalanine (l-DOPA), that 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 Src homology 3 (SH3) domain of human Abelson tyrosine kinase (Abl), 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 Escherichia 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.

Figure 1

Design and application of peptide-based molecular probe expressed in E. coli. Genetically tailored L-DOPA forms covalent linkage to the target protein to alleviate the low affinity of the peptide.

Figure 2

In vivo binding assay using trM2H to evaluate the interaction of TOP1 and BOT1 to the Abl SH3 domain. TetR transcription regulator binds to the Tet-responsive element (TRE) sequence upon dimerization. The binding of dimeric TetR to TRE which is at upstream of the reporter gene (AcGFP) results in the reporter gene expression (Panel iv, positive control). In this system, the dimerization domain of the TetR is replaced with “bait” and “prey”, which are the Abl SH3 domain and TOP1 or BOT1. Positive expression of GFP indicates the interaction of bait and prey.

Figure 3

In vitro characterization of TOP1 and BOT1 interacting with the Abl SH3 domain. (A) Affinity chromatography assay. Fusion proteins GST-TOP1 and GST-BOT1 were immobilized on glutathione sepharose resin and incubated with the E. coli cell lysate expressing MBP-SH3. Unbound proteins were washed off and GST fusion proteins were eluted from the column. Co-purified MBP-SH3 was detected by Western blot probed with anti-MBP antibody. (B) Determination of equilibrium dissociation constant (K_d) between the Abl SH3 domain and TOP1-FITC peptide by measuring fluorescence anisotropy, where max is the anisotropy at the saturation and R is the offset factor.

Figure 4

Expression of the molecular probe TOP1-DOPA-GFP. (A) A schematic diagram of 30.0 kDa TOP1-DOPA-GFP. (B) Total protein analyses of L-DOPA incorporation into TOP1-DOPA-GFP. TOP1-DOPA-GFP was expressed in the presence and absence of L-DOPA, purified with Ni-NTA resin, and resolved by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue. (C) Redox cycling staining of TOP1-DOPA-GFP. Proteins from a similar gel as (B) were blotted to a nitrocellulose membrane and stained with NBT reagent (2 M sodium glycinate, 0.24 mM NBT, pH 10). This method detects quino-proteins and confirmed the presence of L-DOPA/dopaquinone in TOP1-DOPA-GFP.

Figure 5

TOP1-DOPA-GFP in Western blot-type protein analysis. Typical preparation of purified TOP1-DOPA-GFP and wtTOP1-GFP resolved by SDS-PAGE and detected with (A) Coomassie Brilliant Blue staining and (B) redox-cycling staining. (C) SH3-His was resolved by non-denaturing PAGE, blotted to nitrocellulose membranes and probed with TOP1-DOPA-GFP (lanes 1–7) or wtTOP1-GFP (lane 8). NaIO₄ was added to the membranes to oxidize L-DOPA and induce cross-linking between SH3-His and TOP1-DOPA-GFP. (D) Similar gels containing the same amount of SH3-His as (C) were visualized by Coomassie Brilliant Blue staining.