Not all (non-canonical) amino acids are made equal

Global substitution of natural amino acids in proteins with their analogs carrying biorthogonal (biologically inert) functional groups has a variety of applications in basic and applied research. The aim of this study was to apply a previously developed protocol for the incorporation of a methionine analog with a diazirine functional group (photo-methionine, pMet) to a pair of synthetic amino acids with an azido group (azidohomoalanine, Aha) and a triple bond (homopropargylglycine, Hpg). While pMet finds application as a UV-activatable cross-linking agent, the latter two are employed for ligation with other molecules via click chemistry. Although the authors’ aim is to employ all three analogs in an unconventional way to study the electron transfer process between cytochrome b5 and P450, which requires preparing these proteins with high levels of methionine substitution at specific positions.

The growth parameters of E. coli BL-21 and B834 strains used in recombinant expression were studied, and mass spectrometry was used to analyze methionine substitution with its synthetic analogs in three different proteins in a time-resolved manner. A fusion protein construct containing green fluorescent protein (GFP) was chosen for this study for its fluorescence, which allowed for more accurate quantification, and a high amount of methionine in the sequence, which provided information on the site-specific substitution ratio. Although this protein has no application in further experiments, its expression ratio and methionine replacement with analogs was higher than that of cytochrome b5, which is intended for use in future studies. The findings of the study can be used to predict the maximum incorporation ratio for individual analogs provided that the protein of interest is expressed under optimal conditions. Moreover, they can provide clues for revealing optimal conditions. For example, suboptimal conditions can be deduced from elevated levels of the third studied protein, pspA, which plays a role in stress response pathways.