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-benzoylphenylalanine (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.
A p-isothiocyanate phenylalanine mutant of the homodimeric protein homoserine o-succinyltransferase (MetA) was isolated in a temperature dependent selection from a library of metA mutants containing noncanonical amino acids. This mutant protein has a dramatic increase of 24°C in thermal stability compared to the wild type protein. Peptide mapping experiments revealed that the isothiocyanate group forms a thiourea crosslink to the N terminal proline of the other monomer, despite the two positions being >30Å apart in the X-ray crystal structure of the wild type protein. These results show that an expanded set of building blocks beyond the canonical 20 amino acids can lead to significant changes in the properties of proteins.
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