Addition of the keto functional group to the genetic code of Escherichia coli

Abstract

Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E. coli with high translational fidelity in response to the amber nonsense codon. To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative. This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.

Figure 1

SDS/PAGE analysis of Z domain accumulated under different expression conditions. The left lane is a molecular mass marker.

Figure 2

(A) High-resolution FT-ICR mass spectrum of the intact mutant protein Z domain containing p-acetyl-l-phenylalanine. A series of peaks corresponding to different charge states of the protein can be observed. In each series, there are three peaks corresponding to the protein without the first methionine, its acetylated form, and the intact protein as labeled for the 8⁺ charge state. (Inset) The expansion of the molecular peak of the Z domain protein from the 7⁺ isotopic cluster. (B) Tandem mass spectrum of the NH₂-terminal peptide MTSVDNY*INK. The partial sequence of TSVDNY*IN of the peptide containing p-acetyl-l-phenylalanine (Y*) can be assigned from the annotated b (red) and y (blue) ion series.

Figure 3

In vitro labeling of mutant Z domain containing p-acetyl-l-phenylalanine with fluorescein hydrazide 1. (A) Labeling reaction of p-acetyl-l-phenylalanine by fluorescein hydrazide 1. (B) Silver-stained SDS/PAGE (Upper) analysis and fluorescence imaging (Lower) of WT and mutant Z domain labeled with fluorescein hydrazide 1. (C) Fluorescence spectra for WT and mutant Z domain labeled with fluorescein hydrazide 1.

Figure 4

In vitro labeling of mutant Z domain containing p-acetyl-l-phenylalanine with biotin hydrazide 2. (A) The structure of the biotin hydrazide derivative used, 6-({6-[(biotinoyl)amino]hexanoyl}amino)hexanoic acid hydrazide (Molecular Probes). (B) Western blot analysis of WT and mutant Z domain labeled by biotin hydrazide 2.