The Johnson Research Group

Polarization Modulation Studies

The polarization of incoming light is modulated electro-optically. For an immobile fluorophore, absorption of light and thus fluorescence only occurs when its transition dipole aligns with the excitation polarization. This results in modulated fluorescence. A fluorophore that is reorienting on the time scale of the polarization modulation will show little modulation in its fluorescence, and thus the depth of the modulation can be used as a probe of fluorophore mobility.

Polarization graphs of count vs time

 

This technique has been utilized in several experiments involving the binding of CaM to one of its target enzymes, the plasma-membrane calcium ATPase (PMCA). Results suggest heterogeneity in the dynamical structure of the complex of CaM-PMCA complex, an example of the type of information available by single-molecule spectroscopy. The occurrence of occasional periods of low mobility, characterized by high polarization modulation, indicates that the simple picture of CaM bound to a flexible (and therefore orientationally mobile) CaM-binding domain of the PMCA is incomplete, and that an intermediate step exists in the CaM-induced activation of the PMCA (see figure below).

 

Modulation Depth graphs for binding plasma-membrane calcium ATPase

Publications

Osborn, K. D., Zaidi, A., Mandal, A., Urbauer, R. J. B., Johnson, C. K. 2004. Single-Molecule Dynamics of the Calcium-Dependent Activation of Plasma-Membrane Ca2+-ATPase by Calmodulin. Biophysical Journal. 87:1892-1899.

Osborn, K. D., Singh, M. K., Urbauer, R. J. B., Johnson, C. K. 2003. Maximum-likelihood approach to single-molecule polarization modulation analysis. ChemPhysChem. 4:1005-1011.