Amino acid residues 4425-4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor.
We have localized a region contained within the sequence of amino acid residues 4425-4621 on the three-dimensional structure of the skeletal muscle ryanodine receptor (RyR). Mouse monoclonal antibodies raised against a peptide comprising these residues have been complexed with ryanodine receptors and imaged in the frozen-hydrated state by cryoelectron microscopy. These images, along with images of antibody-free ryanodine receptor, were used to compute two-dimensional averaged images and three-dimensional reconstructions. Two-dimensional averages of immunocomplexes in which the ryanodine receptor was in the fourfold symmetrical orientation disclosed four symmetrical regions of density located on the edges of the receptor's cytoplasmic assembly that were absent from control averages of receptor without added antibody. Three-dimensional reconstructions revealed the antibody-binding sites to be on the so-called handle domains of the ryanodine receptor's cytoplasmic assembly, near their junction with the transmembrane assembly. This study is the first to demonstrate epitope mapping on the three-dimensional structure of the ryanodine receptor.
Benacquista
B L
BL
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509, USA. famolaro@wadsworth.org
Sharma
M R
MR
Samsó
M
M
Zorzato
F
F
Treves
S
S
Wagenknecht
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D020285:Cryoelectron Microscopy; D004797:Enzyme-Linked Immunosorbent Assay; D007091:Image Processing, Computer-Assisted; D007074:Immunoglobulin G; D051379:Mice; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000502:physiology*; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel