The resulting force-distance curve (Figure 1) of calmodulin – a calcium-binding protein – reveals that the protein unfolds and refolds in two steps which correspond to two separate helix-loop-helix domains. By simultaneously observing the FRET fluorescence signal, it can be seen how the unfolding of the domains results in fluctuations of the FRET signal due to changes in the relative position of the FRET pair.
Equilibrium dynamics showing the transition between short-lived intermediate states can be studied because of the intrinsic distance clamp which keeps the traps at a fixed distance, while force fluctuations are measured. When this is applied to calmodulin, the equilibrium fluctuations between the states can be observed with a force resolution of <0.1 pN at 100 Hz. Figure 2 shows that calmodulin switches between two major states, without a clear preference and that intermediate steps can be resolved as calmodulin occasionally jumps to a third state for short periods of time, represented by the dashed grey lines.
2 Full length CaM protein at 10 mM Ca2+ showing equilibrium dynamics between multiple states. Data is recorded at 50 kHz (grey line) and averaged at 200 Hz (red line). The histogram quantifies the most populated states in the inset (right panel) showing two peaks at 6.5 ± 0.1 pN and 7.8 ± 0.09 pN (mean ± standard deviation).