Spectroscopic monitoring of local conformational changes during the intramolecular domain-domain interaction of the ryanodine receptor.
The amino (N)-terminal and central regions of the ryanodine receptor (RyR) containing most mutation sites of malignant hyperthermia (MH) and central core disease (CCD) seem to be involved in the Ca(2+) 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 the N-terminal and central domains (zipping) stabilizes the closed-state of the channel, while removal of the contact (unzipping) deblocks the channel, causing channel-activation effects. We here report the results of our recent effort to monitor local conformational changes in the putative domain-domain interaction site 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 peptide corresponding to the Leu(2442)-Pro(2477) region of the central domain) as a site-directing carrier. The site of MCA labeling was localized in the 150 kDa N-terminal region of RyR, indicating that DP4 and its in vivo counterpart (a portion of the central domain) interact with the N-terminal region. RyR-activating domain peptides, DP4 and DP1 (corresponding to the Leu(590)-Cys(609) region of the N-terminal domain), and depolarization of the T-tubule moiety of the triad (physiologic stimulation) induced a rapid decrease in the fluorescence intensity of the protein-bound MCA and Ca(2+) release at a somewhat slower rate. The accessibility of the protein-bound MCA to the fluorescence quencher was increased in the presence of DP4. These results are all consistent with the above hypothesis.
Yamamoto
Takeshi
T
Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Ikemoto
Noriaki
N
D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D008564:Membrane Potentials / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013388:Succinimides / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D013388:Succinimides; C074882:sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate