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Improvements in genetic code expansion have made preparing proteins with diverse functional groups almost routine. Nonetheless, unnatural amino acids (Uaas) pose theoretical burdens on protein solubility, and determinants of position-specific tolerability to Uaas remain underexplored. To broadly examine associations, we systematically assessed the effect of substituting the fluorescent Uaa, acridonylalanine, at more than 50 chemically, evolutionarily, and structurally diverse residues in two bacterial proteins: LexA and RecA. Surprisingly, properties that ostensibly contribute to Uaa tolerability-such as conservation, hydrophobicity, or accessibility-demonstrated no consistent correlations with resulting protein solubility. Instead, solubility is closely dependent on the location of the substitution within the overall tertiary structure, suggesting that intrinsic properties of protein domains, and not individual positions, are stronger determinants of Uaa tolerability. Consequently, those who seek to install Uaas in new target proteins should consider broadening, rather than narrowing, the types of residues screened for Uaa incorporation.

Acridonylalanine (Acd) is a useful fluorophore for studying proteins by fluorescence spectroscopy, but it can potentially be improved by being made longer wavelength or brighter. Here, we report the synthesis of Acd core derivatives and their photophysical characterization. We also performed ab initio calculations of the absorption and emission spectra of Acd derivatives, which agree well with experimental measurements. The amino acid aminoacridonylalanine (Aad) was synthesized in forms appropriate for genetic incorporation and peptide synthesis. We show that Aad is a superior FRET acceptor to Acd in a peptide cleavage assay, and that Aad can be activated by an aminoacyl tRNA synthetase for genetic incorporation. Together, these results show that we can use computation to design enhanced Acd derivatives which can be used in peptides and proteins.

Site-specific fluorescence probes can be used to measure distances within proteins when used as part of a Förster resonance energy transfer (FRET) pair. Here we report the synthesis of a coumarin maleimide (Mcm-Mal) that is fluorogenic upon reaction with cysteine. We demonstrate that cysteine, acridonylalanine (Acd) double mutant proteins can be produced by unnatural amino acid mutagenesis and reacted with Mcm-Mal to generate Mcm/Acd labeled proteins for FRET studies. The Mcm/Acd FRET pair is minimally-perturbing, easy to install, and well-suited to studying protein distances in the 15-40 Å range. Furthermore, Mcm/Acd labeling can be combined with tryptophan fluorescence in three color FRET to monitor multiple interactions in one experiment.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ∼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid.