Mapping the Site of RyR2 “Unzipping” Peptide (DPc10) by using Fluorescence Resonance Energy Transfer (FRET) in Permeabilized Cardiomyocytes

Abstract

DPc10 is a synthetic RyR2 peptide corresponding to the part of central domain (Gly2460-Pro2495) in cardiomyocytes. It was reported that DPc10 can specifically bind and destabilize RyR2 by causing defective domain interaction between N-terminal and central domains, which is called “domain unzipping”. In this study, we use DPc10 as a marker, through small fluorophore and single site specifically labeled proteins, to map the location of DPc10 binding site in permeabilized cardiomyocytes via different FRET pairs. We first test the DPc10 binding stoichiometry, by measuring the ratio of donor (AF488/568-FKBP12.6) enhancement upon acceptor (HF647-DPc10) photobleaching, which show donor and acceptor stoichiometry is 1:1. Two different R0 FRET pairs (AF488/AF568-FKBP12.6 and HF647-DPc10) are used to assess FKBP12.6-DPc10 FRET efficiency in permeabilized cardiomyocytes via acceptor photobleach (PB) and donor quench (Quench).The FRET efficiency between AF568-FKBP12.6 and HF647-DPc10 was 90.8 ± 0.6 % (PB), 91.9 ± 0.6 % (Quench), corresponding to a distance of 55.8 ± 0.6 A (PB), 54.1 ± 0.8 A (Quench). Furthermore, FRET efficiency between AF488-FKBP12.6 and HF647-DPc10 was 57.9 ± 1.4 % (PB), 62.4 ± 1.4 % (Quench), corresponding to the distance of 53.1 ± 0.5 A (PB), 51.4 ± 0.5 A (Quench) which is consistent with the distance measured in FRET between AF568-FKBP12.6 and HF647-DPc10. Moreover, through another FRET pair AF568-CaM and HF647-DPc10, their FRET efficiency was 89.6 ± 0.6 % (PB), which reflects the distance between CaM and DPc10 is 57.2 ± 0.7 A (PB).These results provide direct in situ information of DPc10 localization on the functional RyR2.