Journal
BIOPHYSICAL CHEMISTRY
Volume 193, Issue -, Pages 35-49Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bpc.2014.07.004
Keywords
Fluorescence; Anisotropy; Titration; Intracellular calcium channel; Thermodynamics
Funding
- University of Iowa Center for Biocatalysis and Bioprocessing NIH Biotechnology Training grant [NIH T32 GM08365]
- University of Iowa Carver College of Medicine FUTURE in Biomedicine Fellowship
- National Institutes of Health [R01 GM 57001]
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Calmodulin (CaM) allosterically regulates the homo-tetrameric human Ryanodine Receptor Type 1 (hRyR1): apo CaM activates the channel, while (Ca2+)(4)-CaM inhibits it. CaM-binding RyR1 residues 1975-1999 and 3614-3643 were proposed to allow CaM to bridge adjacent RyR1 subunits. Fluorescence anisotropy titrations monitored the binding of CaM and its domains to peptides encompassing hRyR(11975-1999) or hRyR1(3614-3643). Both CaM and its C-domain associated in a calcium-independent manner with hRyR1(3614-3643) while N-domain required calcium and bound similar to 250-fold more weakly. Association with hRyR1(11975-1999) was weak. Both hRyR1 peptides increased the calcium-binding affinity of both CaM domains, while maintaining differences between them. These energetics support the CaM C-domain association with hRyR1(3614-3643) at low calcium, positioning CaM to respond to calcium efflux. However, the CaM N-domain affinity for hRyR(11975-1999) alone was insufficient to support CaM bridging adjacent RyR1 subunits. Other proteins or elements of the hRyR1 structure must contribute to the energetics of CaM-mediated regulation. (C) 2014 Elsevier B.V. All rights reserved.
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