Advancing Genetically Encoded Lysine (GEK) Chemistry: From Precision Modulation to Therapeutic Innovation

Abstract

ConspectusGenetically encoded lysine (GEK) chemistry has transformed protein engineering by enabling precise and site-specific modifications, which expand lysine's functional landscape beyond its native post-translational modifications (PTMs). Our work has systematically advanced GEK chemistry by developing engineered pyrrolysyl-tRNA synthetase (PylRS) variants that efficiently incorporate diverse lysine (Lys) derivatives with tailored chemical reactivity. By integrating bioorthogonal handles, acyl and electrophilic warheads, photo-cross-linking groups, and PTM mimics, we have established a set of powerful toolkits for protein labeling, functional studies, and Lys-directed drug design. These advances provide precise control over protein structure and function, facilitating the study of epigenetic modifications, enzyme-substrate interactions, and Lys-guided inhibitor development. As GEK chemistry continues to evolve, its integration with structural/synthetic biology and therapeutic innovation will further expand its impact, unlocking new frontiers in chemical biology and precision therapeutics.

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(A) Schematic diagram of incorporation of a ncAA into sfGFP via amber suppression. A ncAA-charged tRNA pairs with the CUA anticodon, enabling full-length fluorescent protein expression; absence of the ncAA leads to truncation. (B) Representative structures of ncAAs (Pyl, BocK, AcK) commonly used in this system.

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(A) Two domains of MmPylRS. (B) Structure of the catalytic core of MmPylRS with Pyl-AMP bound at the active site (PDB entry: 2ZIM). (C) The table of PylRS mutants for Lys-ncAA incorporation. −

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(A) Schematic illustration of SeC incorporation followed by oxidative elimination to generate Dha, enabling subsequent Michael addition. (B) Schematic illustration of AcdK incorporation followed by sequential Staudinger reduction, para-aminobenzyloxycarbonyl self-cleavage, enamine hydrolysis, and reductive amination to yield site-specific Kme2 within proteins. (C) Schematic illustration of PMeK incorporation followed by deprotection into MeK. (D) Genetic incorporation of AznL and site-specific Lys acylation via traceless Staudinger ligation with phosphinothioesters.

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(A) Schematic illustration of KetoK incorporation and condensation reactions of the keto group in genetically encoded ketoK with hydrazides or alkoxyamines. (B) Bioorthogonal dual labeling: incorporated KetoK condenses with coumarin hydroxylamine via oxime formation while AzK reacts with Rhodamine DBCO via SPAAC click reaction. (C) Genetically incorporated AcrK as a versatile chemical handle enabling a range of on-protein reactions including radical polymerization, olefin metathesis, 1,3-dipolar cycloaddition, and S/P-1,4-conjugate addition. (D) HexK incorporation and reaction with tetrazine derivatives via IEDDA reaction. (E) ACTK incorporation and photo-cross-linking with a protein carboxylic group.

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(A) Chemical structure of N ^ε-amino acylated Lys incorporated in our study. (B) ELISA system for site recognition assays of sirtuin enzymes. (C) Sirt6 activities on oc-nucleosomes. (D) SIRT7-catalyzed deacylation activities on eight acyl-nucleosome substrates. The AzHeK residue which could be recognized by Sirt7 was removed and thus was incapable of conjugating DBCO-MB488 dye, resulting lack of fluorescence.

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(A) Schematic diagram of PADLE. (i) ncAAs such as N ^ε-butyryl-l-lysine are genetically incorporated into phage-displayed peptide libraries to anchor ligands at enzymatic active sites. (ii) PADLE biopanning procedure. (B) Chemical structures of PADLE-evolved peptide ligands, each containing a Lys-ncAA (highlighted in blue).