Lysine crotonylation (Kcr) is a histone post-translational modification that is implicated in numerous epigenetic pathways and diseases. Recognition of Kcr by YEATS domains has been proposed to occur through intermolecular amide−π and alkene−π interactions, but little is known about the driving force of these key interactions. Herein, we probed the recognition of lysine crotonylation and acetylation by the AF9 YEATS domain through incorporation of noncanonical Phe analogs with distinct electrostatics at two positions. We found that amide−π interactions between AF9 and acyllysines are electrostatically tunable, with electron-rich rings providing more favorable interactions. This differs from trends in amide−heteroarene interactions and provides insightful information for therapeutic design. Additionally, we report for the first time that CH−π interactions at Phe28 directly contribute to AF9's recognition of acyllysines, illuminating differences among YEATS domains, as this residue is not highly conserved but has been shown to impart selectivity for specific post-translational modification.

The Glaser–Hay bioconjugation has emerged as an efficient method to generate bioconjugates with therapeutic applications.

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.