Single-site glycine-specific labeling of proteins

Abstract

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19 F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.

A single amino acid Gly-tag enables metal-free protein purification

“…S6 †). 11 We were delighted to note that PLP (11i, 50 equiv., 0.1 M NaHCO 3 buffer, pH 7.8) promoted the retro-aldol reaction with 98% conversion within 2 h ( Fig. 5a and Table S1; † for myoglobin and SUMO1, see Fig.…”

“…In this perspective, recent advances in the eld of siteselective protein bioconjugation are encouraging. [6][7][8][9][10][11][12][13][14][15][16][17] However, a chemical technology for the release of the POI under physiological conditions poses the next monumental challenge. This has been the prime reason behind the lack of methods for covalent affinity chromatography (y-z plane, Fig.…”

“…2a). 11 It is noteworthy that the installation of a single Gly residue at the N-terminus of a protein during its expression is convenient. 21 Here, we demonstrate that a single residue (Gly) at the N-terminus can facilitate site-specic immobilization of proteins with a stable C-C bond (step 1, aldol reaction, Fig.…”

Chemical Methods for Selective Labeling of Proteins

“…The capture of latent electrophile generates the five‐membered stable thiazolidine complex (Scheme c) . Recently, we reported the single‐site labeling of N‐terminus Gly (Scheme d) . The method generates an amino alcohol and differentiates N‐Gly in isolated proteins or cell lysates.…”

Fast and Stable N‐Terminal Cysteine Modification through Thiazolidino Boronate Mediated Acyl Transfer

“…Protein modification at the N-terminus offers an appealing alternative [10][11][12][13] as it affords site specificity yet is minimally disruptive to the protein function. In particular, the unique reactivity of N-terminal cysteines (NCys) has been explored, giving rise to the native chemical ligation (NCL) [14] as well as the 2-cyanobenzothiazole (CBT) condensation reactions [15] (Figure 1 a,b).…”