Static and kinetic studies of complex formations between calmodulin and mastoparanX.
Ca(2+)-induced complex formation between calmodulin (CaM) and mastoparanX (MasX) was studied by a fluorescence spectroscopy and by a stopped-flow method. The measurements of the fluorescence anisotropy in the presence of calcium and the fluorescence titration with Ca(2+) revealed that the N- and C-domains of CaM bound cooperatively MasX, while the tryptic fragments of CaM (TR(1)C, 1-77 and TR(2)C, 78-148) bound independently MasX. The Trp-fluorescence stopped-flow experiments revealed that the Ca(2+)-induced binding of CaM and MasX was composed of two processes: one was a rapid binding of the N-domain of CaM to MasX, which was induced by the rapid Ca(2+) binding to the N-sites of CaM. The other was a slow biphasic process. Its fast phase was the binding of the C-domain of CaM to MasX, which was induced by the slow Ca(2+) binding to the C-sites. Interestingly, the kinetics of the slow process varied with the Ca(2+) concentrations. At the low Ca(2+) concentrations, its rate constant increased to around 20 s(-1) as the Ca(2+) concentration increased. At the high Ca(2+) concentrations, the Ca(2+)-induced binding of the C-domain of CaM to MasX proceeded at a constant rate around 20 s(-1). This suggested an existence of a rate-limiting step for the Ca(2+)-induced binding of the C-domain of CaM to MasX at the high Ca(2+) concentrations. The slow phase of the slow process may be a rearrangement of the CaM-MasX complex. These results led to our model of a molecular kinetic mechanism of the Ca(2+)-induced complex formation between CaM and MasX.
Murase
Tomohiko
T
Physics Section, Division of Material Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi, 464-8602, Japan.
Iio
Takayoshi
T
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D003198:Computer Simulation; D005454:Fluorescence Polarization / Q000379:methods; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D017186:Titrimetry; D014357:Trypsin / Q000378:metabolism; D014862:Wasp Venoms / Q000737:chemistry* / Q000378:metabolism*
D002147:Calmodulin; D036341:Intercellular Signaling Peptides and Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D014862:Wasp Venoms; C071123:mastoparan X; D014357:Trypsin; D002118:Calcium