DYNAMICS & STRUCTURE OF INDIVIDUAL CALMODULIN PEPTIDE COMPLEX

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An environmental sensitive probe, Nile Red, tagged to a designed model peptide, was used to probe the structural changes and dynamics of single calmodulin(CaM):peptide complexes. When Ca2+ concentration changes from 0 M to 5 mM, the labeled Nile Red dye shows 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 zero mean and a width of 0.17 for a binning time of 1ms. This finding shows that on the 10 to 100 microsecond 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 Nile Red fluorescence decay rate increase 40% and 180% respectively as the Ca2+ concentration approaches the titration mid-point of 2 uM. These changes are considerably greater than would be expected if the chromophore were either in a homogenous static environment, or in a heterogeneous environment that was exchanging faster than the 1 to 10¿s of second time scale. Thus, PAT analysis appears to be uniquely well suited for studying the presence of and transitions within heterogeneous populations on the micro to second time scale.Such a method should have broad applicability for studying protein folding as well as ligand-protein interactions.