Single-Molecule Dynamics of Conformational Interchange in Calmodulin

The recognition and binding of target binding domains by calmodulin (CaM) require global protein conformational changes. We describe single-molecule measurements of conformational interchange in CaM. CaM labeled with a FRET pair (Alexa Fluor 488 and Texas Red) was trapped in lipid vesicles tethered to the surface of a cover slip. Conformational transitions were observed on the time scale of 2 to 11 ms. The probability of a transition to a compact conformation was significantly lower at low than at high Ca2+ concentration, revealing more frequent transitions to a compact conformational state for CaM with bound Ca2+. These results show that CaM undergoes functional conformational dynamics even in the absence of target enzyme. Conformational searching may permit CaM to readily adopt a binding geometry upon encountering a target binding domain.FRET trajectories were also analyzed by a novel iterative Bayesian propagator to determine the probability distribution of FRET efficiencies as a function of time. We show that this analysis outperforms conventional running averages to resolve conformational jumps and conformational distributions. The figure shows a CaM FRET efficiency probability distribution (color scale). The red line shows the running average. The time axis is in milliseconds.View Large Image | View Hi-Res Image | Download PowerPoint Slide