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Development of inhibitors for histone methyllysine reader proteins is an active area of research due to the importance of reader protein-methyllysine interactions in transcriptional regulation and disease. Optimized peptide-based chemical probes targeting methyllysine readers favor larger alkyllysine residues in place of methyllysine. However, the mechanism by which these larger substituents drive tighter binding is not well understood. This study describes the development of a two-pronged approach combining genetic code expansion (GCE) and structure-activity relationships (SAR) through systematic variation of both the aromatic binding pocket in the protein and the alkyllysine residues in the peptide to probe inhibitor recognition in the CBX5 chromodomain. We demonstrate a novel change in driving force for larger alkyllysines, which weaken cation-π interactions but increases dispersion forces, resulting in tighter binding. This GCE-SAR approach establishes discrete energetic contributions to binding from both ligand and protein, providing a powerful tool to gain mechanistic understanding of SAR trends.

Evolution has converged on cation-π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-π interaction is about -5 kcal mol-1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains.

Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.

A microfluidic device made of polydimethylsiloxane (PDMS) has shown the limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation has minimized the surface-sample contacts and has allowed the in situ addition and combination of other reagents.

The studies have shown that the orthogonal chemistry of a genetically encoded ketone handle together with a single cysteine residue permit efficient and versatile site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer (smFRET) studies. This chemistry is not limited to smFRET labeling and is useful for attachment of other biophysical probes and directed protein immobilization.

The article focuses on a study conducted by researcher M.L. Matthews to demonstrate that a syringomycin halogenase SyrB2 can be used to install nitrogenous anions in place of carbon-hydrogen bonds and generate carbon-nitrogen (C-N) bonds. Topics discussed include promiscuous reactions, addition of a glycine mutation and halogenation activities. The study highlights the limitation of C-N bonds such as low product yield.

A correction to the article "A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo" that was published in the June 23, 2013 issue is presented.