Spectroscopic Monitoring of Local Conformational Changes during the Intramolecular Domain-Domain Interaction of the Ryanodine Receptor

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• 作者：Takeshi Yamamoto and Noriaki Ikemoto
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：February 5, 2002
• 年：2002
• 卷：41
• 期：5
• 页码：1492 - 1501
• 全文大小：193K
• 年卷期：v.41,no.5(February 5, 2002)
• ISSN：1520-4995

文摘

The amino (N)-terminal and central regions of the ryanodine receptor (RyR) containing mostmutation sites of malignant hyperthermia (MH) and central core disease (CCD) seem to be involved inthe Ca²⁺ channel regulation. Our recent peptide probe study (Yamamoto, T., El-Hayek, R., and Ikemoto,N. (2000) J. Biol. Chem. 275, 11618-11625) suggested the hypothesis that a close contact between theN-terminal and central domains (zipping) stabilizes the closed-state of the channel, while removal of thecontact (unzipping) deblocks the channel, causing channel-activation effects. We here report the resultsof our recent effort to monitor local conformational changes in the putative domain-domain interactionsite to test this hypothesis. The conformation-sensitive fluorescence probe, methyl coumarin acetamide(MCA), was incorporated into RyR in a protein- and site-specific manner by using DP4 (the peptidecorresponding to the Leu²⁴⁴²-Pro²⁴⁷⁷ region of the central domain) as a site-directing carrier. The site ofMCA labeling was localized in the 150 kDa N-terminal region of RyR, indicating that DP4 and its invivo counterpart (a portion of the central domain) interact with the N-terminal region. RyR-activatingdomain peptides, DP4 and DP1 (corresponding to the Leu⁵⁹⁰-Cys⁶⁰⁹ region of the N-terminal domain),and depolarization of the T-tubule moiety of the triad (physiologic stimulation) induced a rapid decreasein the fluorescence intensity of the protein-bound MCA and Ca²⁺ release at a somewhat slower rate. Theaccessibility of the protein-bound MCA to the fluorescence quencher was increased in the presence ofDP4. These results are all consistent with the above hypothesis.