Sensor Peptides Based on Fluorescence Resonance Energy Transfer

Sensor peptides based on fluorescence resonance energy transfer (FRET) are described. Prior to developing sensor peptides, FRET-based indicators for the secondary structure of peptides was designed. Helical peptides, AY(KAAAA)nKAC (n = 1, 2, 3), were selected as the model indicator molecules, and the N-terminal α-amino group and C-terminal cysteine side-chain were modified with rhodamine B sulfonyl chloride and fluorescein maleimide, respectively. The resulting fluorescence spectra showed that FRET from fluorescein to rhodamine B was clearly observed in trifluoroethanol (TFE)-containing buffer, and that fluorescence intensity due to the FRET was reduced by addition of guanidine hydrochloride (GuHCl). This is ascribed that the peptide tightly forms a helical structure in TFE, whereas it is stretched by GuHCl. FRET property strongly depends on the distance between fluorescein and rhodamine B, and hence, these FRET behaviors were clearly observed. On the other hand, no significant FRET change of 3mer peptide (AYC) was observed. This is probably due to no distance change, although CD spectrum of AYC altered. Subsequently a FRET-based sensor peptide for a specific peptide was designed. A 10mer sensor peptide (GSYEADRGGC) with a specific affinity to the 12mer peptides was modified with rhodamine B and fluorescein at both terminals, respectively. FRET property was investigated before and after forming a complex with the 12mer peptides as analyte molecules. The resulting fluorescence spectra clearly changed by addition of the analyte peptides. The FRET from fluorescein to rhodamine B was reduced by forming a complex. This is probably due to conformational change and quenching by addition of the analyte peptide. The FRET behavior strongly depended on sequence of the analyte peptides as well as analyte concentration. Furthermore, dissociation constants between the sensor and analyte peptides were estimated to be around 2 x 10-5 M.