Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids

Abstract

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine O-succinyltransferase ( metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with ( p-benzoylphenyl)alanine (pBzF), increases the melting temperature of E. coli metA by 21 °C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Figure 1.. Screening for high thermal stability mutants of metA.

(a) An amber stop codon scanning library of metA was co-transformed with an orthogonal amber suppressor tRNA/aaRS pair into JW3973–1 E. coli. Transformants were incubated in minimal media liquid culture supplemented with 1 mM of the cognate ncAA at 44°C for 20–40 hours. Cells were then plated and colonies were sequenced. The consensus clone F21pBzF metA was retransformed into JW3973–1 and shows an increased growth rate compared to JW3973–1 transformed with WT metA. (b) ncAAs used in this screen.

Figure 2.. Thermal denaturation of metA.

(a) Purified recombinant WT metA (green) and F21pBzF metA (blue) were analyzed by CD spectrometry at 223 nm in 200 mM sodium phosphate, pH 7.4. Fraction unfolded is represented as normalized ellipticity. F21pBzF metA shows a 21°C increase in melting temperature compared to its WT counterpart. (b,c) Mutation of Cys 90 to Ser (b) or Cys 90 alkylation by IA (c) completely reverts the T_m increase of the F21pBzF mutant to WT metA or near WT metA temperatures.

Figure 3.. Expected UAA interaction.

(a) A homology model of E. coli metA was generated based on metA from B. cereus (pdb 2VDJ). The protein is predicted to form a homodimer. The mutant residue, Phe 21 (red), is expected to crosslink with Cys 90 (magenta) of the other monomer. (b) HMBC spectra of ¹³C labeled F21pBzF metA shows non-carbonyl crosspeaks consistent with hemithioketal formation. The 195 ppm carbonyl signal of pBzF was not observed.