High-throughput studies have been widely used to identify protein–protein interactions; however, few of these candidate interactions have been confirmed in vitro. We have used a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observed no interaction between the next enzyme in the pathway, pantothenate synthetase (PS), and aspartate decarboxylase, but did observe an interaction between PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling an adventitiously formed glycine on the protein N-terminal affinity purification tag by using Sortase. Subsequent formation of the protein–protein complex led to apparent restriction of the dynamics of this tag, thus suggesting that this approach could be generally applied to a subset of other protein–protein interaction complexes.

Strain-promoted inverse electron-demand Diels–Alder cycloaddition (SPIEDAC) reactions between 1,2,4,5-tetrazines and strained dienophiles, such as bicyclononynes, are among the fastest bioorthogonal reactions. However, the synthesis of 1,2,4,5-tetrazines is complex and can involve volatile reagents. 1,2,4-Triazines also undergo cycloaddition reactions with acyclic and unstrained dienophiles at elevated temperatures, but their reaction with strained alkynes has not been described. We postulated that 1,2,4-triazines would react with strained alkynes at low temperatures and therefore provide an alternative to the tetrazine cycloaddition reaction for use in in vitro or in vivo labelling experiments. We describe the synthesis of a 1,2,4-triazin-3-ylalanine derivative fully compatible with the fluorenylmethyloxycarbonyl (Fmoc) strategy for peptide synthesis and demonstrate its reaction with strained bicyclononynes at 37 °C with rates comparable to the reaction of azides with the same substrates. The synthetic route to triazinylalanine is readily adaptable to late-stage functionalization of other probe molecules, and the 1,2,4-triazine-SPIEDAC therefore has potential as an alternative to tetrazine cycloaddition for applications in cellular and biochemical studies.

We have investigated the interaction of peptides containing phosphohistidine analogues and their homologues with the prototypical phosphotyrosine binding SH2 domain from the eukaryotic cell signalling protein Grb2 by using a combination of isothermal titration calorimetry and a fluorescence anisotropy competition assay. These investigations demonstrated that the triazole class of phosphohistidine analogues are capable of binding too, suggesting that phosphohistidine could potentially be detected by this class of proteins in vivo.