Investigations of Calmodulin Conformations Resolved by Single Molecule Microscopy

Measurements of the distance between two dye molecules covalently linked to the calcium signaling protein calmodulin (CaM) have been previously performed by our group to investigate the conformations of CaM in solution. It was shown that calmodulin exists in a wide range of distinct conformations whose amplitudes depend upon free calcium concentrations (1). Currently, we are investigating affects that the choice of dye pair or labeling site has on measured conformations. This is done using an alternating laser excitation (ALEX) single molecule microscope system that has been custom built in our laboratory. Time correlated single photon counting in bulk samples is used to determine the time resolved anisotropy of the dye pair and the orientational mobility of each dye. Analysis of burst measurements using interphoton time burst selection criteria and the probability distribution analysis reveal a wide range of CaM conformations. Conformational analysis is performed using both discrete states and the maximum entropy method. The maximum entropy method reveals the most probable underlying conformational distribution that fits our data. Finally, we are investigating fluorescence fluctuations within CaM conformations using conformationally sorted fluorescence correlation spectroscopy.1. Slaughter et al., J. Phys. Chem. B, 2004, 108, 10388-10397