DYNAMICS & STRUCTURE OF INDIVIDUAL CALMODULIN PEPTIDE COMPLEX

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。一种环境敏感的探针，尼罗红，标记到一个设计的模型肽，被用来探测单一钙调素（CaM）：肽复合物的结构变化和动力学。当Ca 2+浓度从0 M变化到5 mM时，标记的尼罗红染料显示荧光强度增加一个数量级。通过将单个钙调素：肽-尼罗红复合物包封在水凝胶中，利用共聚焦显微镜实现了单分子荧光检测。这些单一的复合物获得的荧光偏振分布显示零均值和0.17的宽度为1毫秒的分箱时间。这一发现表明，在10至100微秒的时间尺度上，CaM：肽复合物在水凝胶基质内翻滚。单分子光谱分布提供了由探针感测的极性的异质性的尺度。同时，还得到了钙调素：肽-尼罗红复合物的单分子荧光寿命分布和光子到达时间（PAT）轨迹的钙浓度依赖性。当Ca ~（2+）浓度接近滴定中点2 μ M时，尼罗红荧光衰减速率的均值和方差分别增加40%和180%。如果发色团处于均匀的静态环境中，或者处于交换速度超过1秒到10秒的非均匀环境中，这些变化将大大大于预期。因此，PAT分析似乎是唯一适合于研究的存在和过渡内的异质种群在微米到秒的时间scale.Such方法应该有广泛的适用性，研究蛋白质折叠以及配体-蛋白质相互作用。

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.