FRET-based trilateration of probes bound within functional ryanodine receptors

Bengt Svensson, Tetsuro Oda, Florentin R. Nitu, Yi Yang, Iustin Cornea, Ye Chen-Izu, James D. Fessenden, Donald M. Bers, David D. Thomas, Razvan L. Cornea

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca2+ channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca2+ leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ∼35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

Original languageEnglish (US)
Pages (from-to)2037-2048
Number of pages12
JournalBiophysical journal
Volume107
Issue number9
DOIs
StatePublished - 2014

Bibliographical note

Funding Information:
This work was supported by National Institutes of Health grant No. R01HL092097 (to D.M.B. and R.L.C.), grant No. R01AR059124 (to J.D.F.), and grant Nos. R01GM27906 and R37AG26160 (to D.D.T.).

Publisher Copyright:
© 2014 Biophysical Society.

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