Residue-specific incorporation of non-canonical amino acids into proteins allows facile alteration and enhancement of protein properties. In this review we describe recent technical developments and applications of residue-specific incorporation to problems ranging from elucidation of biochemical mechanisms to engineering of protein-based biomaterials. We hope to inform the reader of the ease and broad utility of residue-specific non-canonical amino acid incorporation with the goal of inspiring investigators outside the field to consider applying this tool to their own research.

Engineering protein translation machinery to incorporate noncanonical amino acids (ncAAs) into proteins has advanced applications ranging from proteomics to single-molecule studies. As applications of ncAAs emerge, efficient ncAA incorporation is crucial to exploiting unique chemistries. We have established a quantitative reporter platform to evaluate ncAA incorporation in response to the TAG (amber) codon in yeast. This yeast display-based reporter utilizes an antibody fragment containing an amber codon at which a ncAA is incorporated when the appropriate orthogonal translation system (OTS) is present. Epitope tags at both termini allow for flow cytometry-based end point readouts of OTS efficiency and fidelity. Using this reporter, we evaluated several factors that influence amber suppression, including the amber codon position and different aminoacyl-tRNA synthetase/tRNA (aaRS/tRNA) pairs. Interestingly, previously described aaRSs that evolved from different parent enzymes to incorporate O-methyl-l-tyrosine exhibit vastly different behavior. Escherichia coli leucyl-tRNA synthetase variants demonstrated efficient incorporation of a range of ncAAs, and we discovered unreported activities of several variants. Compared to a plate reader-based reporter, our assay yields more precise bulk-level measurements while also supporting single-cell readouts compatible with cell sorting. This platform is expected to allow quantitative elucidation of principles dictating efficient stop codon suppression and evolution of next-generation stop codon suppression systems to further enhance genetic code manipulation in eukaryotes. These efforts will improve our understanding of how the genetic code can be further evolved while expanding the range of chemical diversity available in proteins for applications ranging from fundamental epigenetics studies to engineering new classes of therapeutics.

The reactions of acetic acid, acetic-d3 acid-d, and formic acid with the Ge(100)-2 x 1 surface have been investigated using multiple internal reflection Fourier transform infrared (MIR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and density functional theory (DFT) calculations. The infrared and photoelectron data provide experimental evidence for an O-H dissociation product at 310 K. DFT calculations indicate that the O-H dissociation pathway is significantly favored, both kinetically and thermodynamically, over other potential reaction pathways. All of the carboxylic acids studied exhibit unexpected vibrational modes between 1400 and 1525 cm(-1), which are attributed to the presence of a bidentate bridging structure where both oxygen atoms interact directly with the surface.

Yeast surface display is a powerful technology for engineering a broad range of protein scaffolds. This protocol describes the process for de novo isolation of protein binders from large combinatorial libraries displayed on yeast by using magnetic bead separation followed by flow cytometry-based selection. The biophysical properties of isolated single clones are subsequently characterized, and desired properties are further enhanced through successive rounds of mutagenesis and flow cytometry selections, resulting in protein binders with increased stability, affinity, and specificity for target proteins of interest.