Probing Structural Dynamics of Individual Calmodulin:Peptide Complexes in Hydrogels by Single-Molecule Confocal Microscopy

An environment-sensitive probe, Nile Red, tagged to a designed model peptide, is used to probe the structural changes and dynamics of single calmodulin (CaM):peptide complexes. When the Ca 2+ concentration changes from 0 M to 5 mM, the labeled Nile Red dye undergoes an order of magnitude increase in fluorescence intensity. By encapsulation of single CaM:peptide−Nile Red complexes into a hydrogel, single-molecule fluorescence detection has been achieved using confocal microcopy. The fluorescence polarization distribution obtained for these single complexes shows a mean of 0 and a width of 0.17, for a binning time of 1 ms. This finding shows that on the 10−100-μs time scale the CaM:peptide complex is tumbling within the hydrogel matrix. The single-molecule spectral distribution provides the scale of the heterogeneity of the polarity sensed by the probe. The calcium concentration dependency of the single-molecule-fluorescence lifetime distributions and photon-arrival-time (PAT) trajectories of the CaM:peptide−Nile Red complexes were also obtained. The mean and variance of the Nile Red fluorescence decay rate increase 40% and 180%, respectively, as the Ca 2+ concentration approaches the titration midpoint of 2 μM. These changes are considerably greater than would be expected if the chromophore was in a homogeneous static environment, or in a heterogeneous environment that was exchanging faster than a time scale of 1 s to 10's of seconds. Thus, PAT analysis appears to be uniquely well suited to study the dynamics of the peptide dissociation from the CaM:peptide complex in the presence of [Ca 2+ ] and transitions within heterogeneous populations on the microsecond−second time scale.