Effect of macrocyclization and TAMRA-labeling on chemokine binding peptides.
Julia S WackKevin BrahmPhilipp BabelJames A R DaltonKatja SchmitzPublished in: Journal of peptide science : an official publication of the European Peptide Society (2023)
Receptor-derived peptides have played an important role in elucidating chemokine-receptor interactions. For the inflammatory chemokine CXCL8, a site II-mimetic peptide has been derived from parts of extracellular loops 2 and 3 and adjacent transmembrane helices of its receptor CXCR1 (Helmer et al., RSC Adv., 2015, 5, 25657). The peptide sequence with a C-terminal glutamine did not bind to CXCL8, whereas one with a C-terminal glutamate did but with low micromolar affinity. We sought to improve the affinity and protease stability of the latter peptide through cyclization whilst also cyclizing the former for control purposes. To identify a cyclization strategy that permits a receptor-like interaction, we conducted a molecular dynamics (MD) simulation of CXCL8 in complex with full-length CXCR1. We introduced a linker to provide an appropriate spacing between the termini and used an on-resin side chain-to-tail cyclization strategy. Upon chemokine binding, the fluorescence intensity of the tetramethylrhodamine (TAMRA)-labeled cyclic peptides increased while the fluorescence anisotropy decreased. Additional MD simulations indicated that the fluorophore interacts with the peptide macrocycle so that chemokine binding leads to its displacement and observed changes in fluorescence. Macrocyclization of both 18-amino acid-long peptides led to the same low micromolar affinity for CXCL8. Likewise, both TAMRA-labeled linear peptides interacted with CXCL8 with similar affinities. Interestingly, the linear TAMRA-labeled peptides were more resistant to tryptic digestion than the unlabeled counterparts, whereas the cyclized peptides were not degraded at all. We conclude that the TAMRA fluorophore tends to interact with peptides altering their protease stability and behavior in fluorescence-based assays.