Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy.
We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.
Liu
Ruchuan
R
Fundamental Science Directorate, Chemical Science Division, Pacific Northwest National Laboratory, P.O. Box 999, MSIN K8-88, Richland, Washington 99352, USA.
Hu
Dehong
D
Tan
Xin
X
Lu
H Peter
HP
D002147:Calmodulin / Q000737:chemistry*; D008958:Models, Molecular; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods*
D002147:Calmodulin; D011506:Proteins