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

Abstract

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 Ca2+ 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 Ca2+ 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 [Ca2+] and transitions within heterogeneous populations on the microsecond−second time scale.

Single-Molecule Protein Interaction Conformational Dynamics

“…However, a large A indicates a more reliable fitting of the kinetics. Dynamics at the sub-millisecond and millisecond time-scale were also reported for apo-CaM, Ca 2+ -CaM, and the CaM-peptide using single-molecule spectroscopy [30,31]. The dynamics in fluorescence polarization revealed by autocorrelation functions imply the presence of considerable heterogeneity in the rotation of the CaM peptide due to conformational change.…”

Role of Calcium in Modulating the Conformational Landscape and Peptide Binding Induced Closing of Calmodulin

“…This indicates that calcium enhances the inherent flexibility of the linker. This flexibility of apo-CAM and Ca-CAM has been explored previously by using both experimental and computational techniques. − This flexibility in proteins is important as it allows them to access functionally relevant conformations. − In the same article by Vendrusculo and co-workers, another ensemble of 160 structures (PDB ID 2K0F) of calcium-loaded calmodulin (Ca-CAM) bound to a small 19-residue helical peptide has been reported . This peptide is derived from smooth muscle myosin light chain kinase (smMLCK).…”

Exploring and Engineering the Conformational Landscape of Calmodulin through Specific Interactions

“…Small-angle X-ray scattering and NMR studies also admit substantial conformational plasticity in CaM in terms of interdomain distance. Despite numerous experimental and simulation studies regarding the conformational flexibility of CaM, the molecular origin behind such conformational flexibility is yet to be resolved. ,,− …”