Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus.
Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [(3)H]ryanodine binding that was sensitive to activation by Ca(2+) and caffeine and to inhibition by Mg(2+). 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the "clamp" structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.
Liu
Z
Z
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, USA. liuz@wadsworth.org
Zhang
J
J
Sharma
M R
MR
Li
P
P
Chen
S R
SR
Wagenknecht
T
T
D002460:Cell Line; D020285:Cryoelectron Microscopy / Q000379:methods; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology / Q000648:ultrastructure; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology / Q000648:ultrastructure*
D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D005982:Glutathione Transferase