Site-specific protein labeling using PRIME and chelation-assisted click chemistry

Abstract

This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: PRobe Incorporation Mediated by Enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase site-specifically attaches a picolyl azide derivative to a 13-amino acid recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing picolyl azide are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize picolyl azide, perform PRIME labeling, and achieve CuAAC derivatization of picolyl azide on live cells, fixed cells, and purified proteins. Reagent preparations, including synthesis of picolyl azide probes and expression of lipoic acid ligase, take 12 d, while the procedure to perform site-specific picolyl azide ligation and CuAAC on cells or on purified proteins takes 40 min-3 h.

A Mechanical Switch Couples T Cell Receptor Triggering to the Cytoplasmic Juxtamembrane Regions of CD3ζζ

“…Testing our hypothesis required resolving intra- (cis) from inter-module (trans) interactions between CD3ζζ subunits. We addressed this directly through the use of a probe ligase (the W37V variant of E. coli lipoic acid ligase (LplA W37V )) in combination with its peptide substrate (ligase acceptor peptide 1 or LAP1) for Interaction-Dependent PRobe Incorporation Mediated by Enzymes (ID-PRIME) ( Figure S2A, S3A-S3C )(Slavoff et al, 2011; Uttamapinant et al, 2013; Uttamapinant et al, 2010). The affinity between LplA W37V and LAP1 has been tuned such that probe transfer is efficient only when ligase and peptide are proximal, thus providing fine spatial resolution in live cells.…”

A Fluorimetric Readout Reporting the Kinetics of Nucleotide‐Induced Human Ribonucleotide Reductase Oligomerization

“…However, because ATTO dye labeling of the His 6 tag is noncovalent, we observed dye dissociation under dilute reaction conditions. We also attempted to use a Lap tag, [20] which can be enzymatically modified, inducing covalent labeling of the Lap-tagged protein. However, the N-terminal Lap-tagged-α failed to hexamerize upon treatment with hexamerization inducers.…”

Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation

“…Chelation-assisted copper catalysis is a recent and complementary approach to ligand-based acceleration (Brotherton et al, 2009; Kuang et al, 2011; Kuang et al, 2010), designed to enhance the “weakest link” in the CuAAC mechanism: the association of azide with the metal center (Rodionov et al, 2007a). For example, a bidentate picolyl azide (shown in Figure 4D) was used in combination with BTTAA for site-specific conjugations of proteins on the surface of live cells with metabolically labeled RNA and protein molecules, providing 25-fold enhancement in specific labeling rate relative to conventional non-chelating azides at low copper concentrations (10–100μM) (Jiang et al, 2014; Uttamapinant et al, 2013; Uttamapinant et al, 2012). Azides bearing tighter binding ligands such as tetradentate bis(triazolyl)amino azide, have also exhibited outstanding reactivity with alkynes under dilute conditions in the presence of complex cellular media, and was used for the tracking of paclitaxel inside living cells (Bevilacqua et al, 2014).…”