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Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus.

Z. Liu, J. Zhang, M. Sharma, P. Li, S. Chen, and T. Wagenknecht. Proc. Natl. Acad. Sci. U. S. A. 98 (11): 6104--6109 (May 2001)

Abstract

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)Hryanodine 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.

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DOI:
10.1073/pnas.111382798
URL:
http://dx.doi.org/10.1073/pnas.111382798
BibTeX key:
Liu_2001_6104
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