Revealing Two-State Protein-Protein Interactions of Calmodulin by Single-Molecule Spectroscopy

Journal Article · Tue Aug 01 00:00:00 EDT 2006 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/ja057005m· OSTI ID:1152379

Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H. Peter

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. Finally, 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.

• Research Organization: Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• OSTI ID: 1152379
• Journal Information: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Journal Issue: 31 Vol. 128; ISSN 0002-7863
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English