In situ monitoring of conformational changes of and peptide bindings to calmodulin on a 27 MHz quartz-crystal microbalance.
Conformational changes of calmodulin (CaM) by additions of Ca(2+) ions and bindings of CaM-binding peptides to Ca(2+)/CaM followed by conformational changes were monitored by a CaM-immobilized 27 MHz quartz-crystal microbalance (QCM) with an admittance analysis. Both the binding and the conformational change events could be detected from the time-dependence of frequency decreases (mass increases) and energy dissipation decreases (elasticity increases), respectively. When Ca(2+) ions were injected to a QCM cell on which biotinylated CaM was immobilized with avidin-biotin interactions, a frequency increase (a mass decrease) and an energy dissipation decrease (an elasticity increase) were observed because of the dehydration and the elasticity increase caused by conformational changes from the flexible CaM to the rigid Ca(2+)/CaM exposing the hydrophobic surface. In the case of the addition of CaMKII-peptide in the Ca(2+)/CaM-immobilized QCM, the immediate frequency decrease (the mass increase) due to the binding of the peptide to Ca(2+)/CaM and the following energy dissipation decrease (the elasticity increase) with a time lag were observed. This suggests that the interaction of the CaMKII-peptide to Ca(2+)/CaM follows an allosteric binding mode. Binding kinetics of the peptide to Ca(2+)/CaM (k(1) and k(-1)) and kinetics of the following conformational change of Ca(2+)/CaM (k(2) and k(-2)) could be obtained. This technique is useful to investigate biomolecular interactions involving the conformational and/or viscoelastic property changes that are biologically important.
Furusawa
Hiroyuki
H
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan.
Komatsu
Mayu
M
Okahata
Yoshio
Y
D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry*; D003460:Crystallization / Q000379:methods; D004548:Elasticity; D036103:Nanotechnology; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011791:Quartz / Q000737:chemistry*
D002147:Calmodulin; D010455:Peptides; D011791:Quartz