Probing the polarity and water environment at the protein-peptide binding interface using tryptophan analogues.

7-Azatryptophan and 2,7-diazatryptophan are sensitive to polarity changes and water content, respectively, and should be ideal for studying protein-protein and protein-peptide interactions. In this study, we replaced the tryptophan in peptide (LKWKKLLKLLKKLLKLG-NH) with 7-azatryptophan or 2,7-diazatryptophan, forming (7-aza)Trp- and (2,7-aza)Trp, to study the calmodulin ()-peptide interaction. Dramatic differences in the (7-aza)Trp- and (2,7-aza)Trp fluorescence properties between free peptide in water and calmodulin-bound peptide were observed, showing a less polar and water scant environment at the binding interface of the peptide upon calmodulin binding. The affinity of the peptides for binding followed the trend (210±10 pM)<(7-aza)Trp- (109±5 pM)<(2,7-azaTrp (45±2 pM), showing moderate increase in binding affinity upon increasing the number of nitrogen atoms in the Trp analogue. The increased binding affinity may be due to the formation of more hydrogen bonds upon binding for the Trp analogue with more nitrogen atoms. Importantly, the results demonstrate that (7-aza)Trp and (2,7-aza)Trp are excellent probes for exploring the environment at the interface of protein-peptide interactions.