It has been shown that the activity of RyR1 is regulated by oxidation (37), and this enzyme activity might in some manner control redox status and hence activity of the channel. Conclusion In conclusion, we have used computational methods to predict a structure and function for one domain of RyR1. of events that lead to muscle contraction. RyR1 is a homotetramer (1) whose subunits are 565 kDa (e.g., human, 5,038 residues; rabbit, 5,037 residues) (2, 3). Mutations in three domains of this protein, one of which is between amino acids 35 and 614, have been implicated in the pathogenesis of two human diseases, malignant hyperthermia and central core disease (4, 5). The Ca2+ release channel exists in at least two functional states, opened and closed (6), which likely have conformational differences. The low-resolution structures of the Ca2+ release channel in different functional states have been studied extensively by electron cryomicroscopy (7C9). On opening, a number of structural changes occur in several regions of the channel, including both the clamp-like domains in the cytoplasmic CD127 region and the transmembrane domain. The clamp domains are the most likely candidates for interaction with DHPR (8) and must, therefore, be allosterically coupled to the transmembrane domain in order for DHPR to induce the opening of the Ca2+ permeable pore of RyR1. Here we describe a unique approach for identification of new functional and structural domains of this complex protein. Methods Sequence Analysis. Initial motif searching in the primary sequence of rabbit RyR1 (P11716) was done by using proscan (10) with a threshold of 70%. Subsequently, 500-residue consecutive, serial sequence segments of the RyR1 were submitted to the University of California, Los AngelesCDepartment of Energy (UCLACDOE) Fold recognition server (11). Primary sequence alignments were performed by using CLUSTALW (Gonnet weight matrix) with a Gonnet Pam250 positive-value similarities scoring system (12, 13). Additionally, multiple sequence alignments were done with additional RyR sequences. As sequence identity in this region is extremely high, only rabbit RyR1 is definitely shown. Sample Preparation. RyR1 was purified from your rabbit skeletal muscle mass SR membranes as explained (14). Electron Cryomicroscopy and Image Control. Purified RyR1 in the closed conformation in the presence of 1 mM EGTA 2-Naphthol (free Ca2+ 10 nM) was prepared for electron cryomicroscopy (15) and examined inside a JEOL1200 electron microscope managed at 100 kV (9). Images were recorded on Kodak SO-163 film at a nominal magnification of 40,000. The micrographs were digitized by using a Zeiss SCAI scanner with a step size of 14 m. A total of 7,300 particle images were selected from 10 micrographs. Solitary particle reconstruction with total amplitude and phase correction of the contrast transfer function was performed in EMAN (16). A resolution of 22 ? using the standard 0.5 Fourier shell correlation criterion was calculated. Note that our earlier publications (7C9) used the 3 resolution criteria which would have yielded 19 ?. Homology Modeling. A homology model of the N-terminal website of RyR1 (residues 41C420) was constructed in INSIGHTII with the Homology and Modeler packages (Accelrys, San Diego) using 4ICD and three related oxidoreductases, 9ICD (18), 1IDE (19), and 1GRO (20). rms deviation (rmsd) between the homology models and templates were determined ( 1 ? rmsd) using the magic fit option in the SWISSPDB Audience (21). A 20 ? resolution denseness model (1283 voxel map, 5.25 ? per pixel) of the homology modeled website was created by using (16). Collapse Localization. FOLDHUNTER was initially run to localize the modeled website to RYR1 with an angular step size of 10, where a minimum of 12 is required for accurate localization at 22 ?. A refinement of the FOLDHUNTER, using the intelligent option, was carried out in a section related to one of the four equal subunits of 2-Naphthol the closed state 2-Naphthol structure. Visualization of the fitted was done by using IRIS EXPLORER (NAG, Downers Grove, IL). Antibody Labeling and Difference Imaging. The sequence-specific antibody against synthetic peptide with sequence KGLDSFSGKPRGSGPPAGP related to residues 416C434 of the RyR1 coupled to keyhole limpet hemocyanin was produced in rabbits by Pel Freeze Biologicals (Rogers, AR). The antibody was purified using a protein A affinity column (Pierce Endogen) relating to manufacturer’s protocol and an antigenic peptide-affinity column (22). The antibodies were characterized by ELISA assays and Western blotting analysis with SR membranes and purified RyR1. RyR1/antibody immunocomplexes were prepared by incubating purified RyR1 (0.2 mg/ml) with purified IgG (0.1 mg/ml) at 1:12 molar percentage in the presence of EGTA, to minimize possible practical transitions of.
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