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Papers In Press, published online ahead of print September 24, 2002
Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509
Corresponding Author: liuz{at}wadsworth.org
Summary Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2wt), and the other encoding modified RyR2, into which was inserted a cDNA encoding Green Fluorescent Protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2D4365-GFP). HEK293 cells expressing both RyR2wt and RyR2D4365-GFP cDNAs showed caffeine- and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2D4365-GFP, were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryo-electron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional (3D) reconstructions were obtained by single particle image processing. The 3D reconstruction of RyR2wt appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the 3D structure of RyR2 to one of the subunit’s characteristic domains, domain 3, also known as the “handle” domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the 3D structure of RyR.
J. Biol. Chem, 10.1074/jbc.M208124200
Submitted on August 8, 2002
Revised on September 17, 2002
Accepted on September 24, 2002
Three-dimensional reconstruction of the recombinant type 2 ryanodine receptor and localization of its divergent region 1
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