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.
%0 Journal Article
%1 Liu_2001_6104
%A Liu, Z.
%A Zhang, J.
%A Sharma, M. R.
%A Li, P.
%A Chen, S. R.
%A Wagenknecht, T.
%D 2001
%J Proc. Natl. Acad. Sci. U. S. A.
%K 11353864 Acid, Adenosine Alanine, Animals, Caffeine, Calcium Calcium, Cell Channel Channel, Co, Conformation, Cryoelectron Dose-Response Drug, Electrophysiology, Fusion Gating, Gl, Glutamic Glutathione Gov't, Heart, Humans, Ion Line, Magnesium, Mice, Microscopy, Muscle Mutation, Non-U.S. P.H.S., Point Protein Proteins, Receptor Recombinant Relationship, Release Research Reticulum, Ryanodine Ryanodine, Sarcoplasmic Support, Transfection, Transferase, Triphosphate, U.S. ntraction, utamic
%N 11
%P 6104--6109
%R 10.1073/pnas.111382798
%T Three-dimensional reconstruction of the recombinant type 3 ryanodine
receptor and localization of its amino terminus.
%U http://dx.doi.org/10.1073/pnas.111382798
%V 98
%X 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.
@article{Liu_2001_6104,
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 c{DNA}s
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 {C}a$^{2+}$
and caffeine and to inhibition by {M}g$^{2+}$. 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.},
added-at = {2009-06-03T11:20:58.000+0200},
author = {Liu, Z. and Zhang, J. and Sharma, M. R. and Li, P. and Chen, S. R. and Wagenknecht, T.},
biburl = {https://www.bibsonomy.org/bibtex/20a1e5bdb2a6cef954ce4aca06a2a4862/hake},
description = {The whole bibliography file I use.},
doi = {10.1073/pnas.111382798},
file = {Liu_2001_6104.pdf:Liu_2001_6104.pdf:PDF},
interhash = {ea198676702a3cec4707da9fcf89eb46},
intrahash = {0a1e5bdb2a6cef954ce4aca06a2a4862},
journal = {Proc. Natl. Acad. Sci. U. S. A.},
keywords = {11353864 Acid, Adenosine Alanine, Animals, Caffeine, Calcium Calcium, Cell Channel Channel, Co, Conformation, Cryoelectron Dose-Response Drug, Electrophysiology, Fusion Gating, Gl, Glutamic Glutathione Gov't, Heart, Humans, Ion Line, Magnesium, Mice, Microscopy, Muscle Mutation, Non-U.S. P.H.S., Point Protein Proteins, Receptor Recombinant Relationship, Release Research Reticulum, Ryanodine Ryanodine, Sarcoplasmic Support, Transfection, Transferase, Triphosphate, U.S. ntraction, utamic},
month = May,
number = 11,
pages = {6104--6109},
pii = {111382798},
pmid = {11353864},
timestamp = {2009-06-03T11:21:20.000+0200},
title = {Three-dimensional reconstruction of the recombinant type 3 ryanodine
receptor and localization of its amino terminus.},
url = {http://dx.doi.org/10.1073/pnas.111382798},
volume = 98,
year = 2001
}