In Situ Monitoring of Conformational Changes of and Peptide Bindings to Calmodulin on a 27 MHz Quartz-Crystal Microbalance

• Published in: Analytical chemistry (Washington) Vol. 81; no. 5; pp. 1841 - 1847
• Main Authors: Furusawa, Hiroyuki, Komatsu, Mayu, Okahata, Yoshio
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.03.2009
• Subjects: Analytical chemistry; Binding Sites; Calmodulin - chemistry; Chemical bonds; Chemistry; Crystallization - methods; Elasticity; Exact sciences and technology; General, instrumentation; Ions; Nanotechnology; Peptides; Peptides - chemistry; Protein Binding; Protein Conformation; Quartz; Quartz - chemistry; Reaction kinetics; Time dependence; Peptides; In situ; Immobilization; Hydrophobic compound; Dehydration; Conformational changes; Calmodulin; Monitoring; Quartz microbalance
• DOI: 10.1021/ac8022229
• ISSN: 0003-2700

Conformational changes of calmodulin (CaM) by additions of Ca2+ ions and bindings of CaM-binding peptides to Ca2+/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 Ca2+ 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 Ca2+/CaM exposing the hydrophobic surface. In the case of the addition of CaMKII-peptide in the Ca2+/CaM-immobilized QCM, the immediate frequency decrease (the mass increase) due to the binding of the peptide to Ca2+/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 Ca2+/CaM follows an allosteric binding mode. Binding kinetics of the peptide to Ca2+/CaM (k 1 and k −1) and kinetics of the following conformational change of Ca2+/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.