title: FRET-based trilateration of probes bound within functional ryanodine receptors.
abstract: To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) 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 Ca(2+) 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.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota.
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.affiliation: Department of Pharmacology, University of California, Davis, California.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Oda
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota.
authors.forename: Florentin R
authors.initials: FR
authors.lastname: Nitu
authors.affiliation: Department of Pharmacology, University of California, Davis, California.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota.
authors.forename: Iustin
authors.initials: I
authors.lastname: Cornea
authors.affiliation: Department of Pharmacology, University of California, Davis, California.
authors.forename: Ye
authors.initials: Y
authors.lastname: Chen-Izu
authors.affiliation: Department of Anesthesia, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
authors.forename: James D
authors.initials: JD
authors.lastname: Fessenden
authors.affiliation: Department of Pharmacology, University of California, Davis, California.
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota. Electronic address: corne002@umn.edu.
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
mesh_terms: D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry; D003198:Computer Simulation; D020285:Cryoelectron Microscopy / Q000379:methods*; D003461:Crystallography / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D015394:Molecular Structure; D032383:Myocytes, Cardiac / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; C451408:ryanodine receptor calcium release channel (2460-2495), synthetic; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B