Expanding Genetic Code for Protein Lysine and Phenylalanine Modifications
Abstract
The naturally occurring pyrrolysine (Pyl) incorporation machinery was discovered in methanogenic archaea and some bacteria. In these organisms, Pyl is cotranslationally inserted into proteins and coded by an in-frame UAG codon. Suppression of this UAG codon is mediated by a suppressor tRNA, (tRNA_CUA)^Pyl , that has a CUA anticodon and is acylated with Pyl by pyrrolysyl-tRNA synthetase (PylRS). The PylRS-(tRNA_CUA)^Pyl pair can be directly applied to incorporate Pyl and other lysine derivatives into proteins at amber mutation sites in E. coli and mammalian cells. In the approach of amber codon suppression, evolved PylRSs were selected to synthesize the proteins genetically with lysine and phenylalanine derivatives which contain native or mimic of post-translational modifications (PTMs) or active chemical functional groups for protein labelling and protein folding studies.
A photocaged N^6-methyl-L-lysine has been genetically incorporated into proteins at amber codons in Escherichia coli using an evovled PylRS-(tRNA_CUA)^Pyl pair. Its genetic incorporation and following photolysis to recover N?-methyl-L-lysine at phsyiological pH provide a convenient method for the biosythesis of proteins with monomethylated lysines. Using an evolved PylRS-(tRNA_CUA)^Pyl pair, a Se-alkylselenocysteine was genetically incorporated in histone H3. The H3 with mimics of PTMs such as lysine methylation, lysine acetylation, and serine phosphorylation has been synthesized by selective oxidative elimination of Se-alkylselenocysteine and followed Michael addition reactions with different thiol-containing small molecules.
Using evolved PylRS -(tRNA_CUA)^Pyl pairs, L-phenylalanine, p-iodo-L-phenylalanine and p-bromo-L-phenylalanine have been genetically incorporated into proteins at amber mutation sites in E. coli. The drastic change of the substrate specificity of PylRS from an aliphatic amino acid to short aromatic amino acids indicates that the PylRS-(tRNA_CUA)^Pyl pair can be evolved for genetic incorporation of a large variety of NAAs into proteins in E. coli. Inspired by the consistent mutations on N346 position, the mutants on N346 and C348 were constructed and evaluated with different L-phenylalanine derivatives. Using PylRS - N346A/C348A (tRNA_CUA)^Pyl pair, more than 30 L-phenylalanine derivatives have been genetically incorporated into proteins at defined sites with amber mutation in E. coli. These breakthroughs and development greatly expand the inventory of genetically encoded NAAs and our abilities to do protein engineering in these cells.
Subject
Protein labelingProtein folding
Histones
Protein post-translational modifications
Amber codon suppression
Pyrrolysyl-tRNA synthetase
Citation
Wang, Yane-Shih 1977- (2012). Expanding Genetic Code for Protein Lysine and Phenylalanine Modifications. Doctoral dissertation, Texas A & M University. Available electronically from https : / /hdl .handle .net /1969 .1 /ETD -TAMU -2012 -08 -11542.