Genetically Encoded Quinone Methides Enabling Rapid, Site-Specific, and Photocontrolled Protein Modification with Amine Reagents

Abstract

Site-specific modification of proteins with functional molecules provides powerful tools for researching and engineering proteins. Here we report a new chemical conjugation method which photocages highly reactive but chemically selective moieties, enabling the use of protein-inert amines for selective protein modification. New amino acids FnbY and FmnbY, bearing photocaged quinone methides (QMs), were genetically incorporated into proteins. Upon light activation, they generated highly reactive QM, which rapidly reacted with amine derivatives. This method features a rare combination of desired properties including fast kinetics, small and stable linkage, compatibility with low temperature, photocontrollability, and widely available reagents. Moreover, labeling via FnbY occurs on the β-carbon, affording the shortest linkage to protein backbone which is essential for advanced studies involving orientation and distance. We installed various functionalities onto proteins and attached a spin label as close as possible to the protein backbone, achieving high resolution in double electron−electron paramagnetic resonance distance measurements.

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction

“…Therefore, a labeling method with short linker will enrich the labeling toolbox and may benefit smFRET measurement under certain circumstance. 35 In this work, we demonstrate the highly efficient and specific genetic incorporation of the novel unnatural amino acid, 2-amino-3-(4-hydroselenophenyl) propanoic acid, or seleno-phenylalanine (SeF) (Fig. 2G), through genetic code expansion, followed by Se-Click reaction to conjugate the Bodipy593 fluorophore on proteins (Fig.…”

New covalent bonding ability for proteins

“…Through GST dimeric cross‐linking, nine amino acid residues with nucleophilic side chains (Cys, Lys, His, Tyr, Trp, Met, Arg, Asn, and Gln) in close proximity were found to covalently linked with the photoactivated FnbY 26 and FmnbY (unpublished results). What is more, when there is no nucleophilic residue nearby, the photoactivated FnbY and FmnbY in proteins also allow rapid (<10 s) conjugation of amine or thiol reagents 27 …”

“…We demonstrated that fluoroacetamide, installed on Uaa FAcK (Figure 2a) and previously thought inert in cells, actually reacted with the thiol group of cysteine when in proximity. Introducing the functional group into testing proteins as a Uaa cannot always be successful, so a facile method to attach functionalities onto proteins is later developed 27 . Uaa FnbY and FmnbY are genetically incorporated into proteins at the desired site, which generate QM upon UV activation (Figure 2d).…”

Genetic Incorporation of ϵ‐N‐Benzoyllysine by Engineering Methanomethylophilus alvus Pyrrolysyl‐tRNA Synthetase

“…MaPylRS is also more stable and has higher expression level in E. coli cells [28] . Transplantation of mutations identified in the active sites of MmPylRS or MbPylRS into the MaPylRS often lead to improved ncAA incorporation efficiency in E. coli [29,30] . MaPylRS protein can also be concentrated several fold higher without aggregation in vitro , which greatly facilitates cell‐free genetic code expansion as well [28] …”