Pyrene-Apelin Conjugation Modulates Fluorophore- and Peptide-Micelle Interactions.

ABSTRACT

Bioactive apelin peptide forms ranging in length from 12 to 55 amino acids bind to and activate the apelin receptor (AR or APJ), a class A G-protein coupled receptor. Apelin-12, -17, and -36 isoforms, named according to length, with an additional N-terminal cysteine residue allowed for regiospecific and efficient conjugation of pyrene maleimide. Through steady-state fluorescence spectroscopy, the emission properties of pyrene in aqueous buffer were compared to those of the pyrene-apelin conjugates both without and with zwitterionic or anionic micelles. Pyrene photophysics are consistent with an expected partitioning into the hydrophobic micellar cores, while pyrene-apelin conjugation prevented this partitioning. Apelin, conversely, is expected to preferentially interact with anionic micelles; pyrene-apelin conjugates appear to lose preferential interaction. Finally, Förster resonance energy transfer between pyrene and tryptophan residues in the N-terminal tail and first transmembrane segment (the AR55 construct, comprising residues 1-55 of the AR) was consistent with efficient nonspecific pyrene-apelin conjugate binding to micelles rather than direct, specific apelin-AR55 binding. This approach provides a versatile fluorophore conjugation strategy for apelin, particularly valuable given that even a highly hydrophobic fluorophore is not deleterious to peptide behavior in membrane-mimetic micellar systems.