Investigating the landscape of intracellular [Ca] in live cells by rapid photoactivated cross-linking of calmodulin-protein interactions.

The Ca sensor protein calmodulin interacts in a Ca-dependent manner with a large number of proteins that among them encompass a diverse assortment of functions and subcellular localizations. A method for monitoring calmodulin-protein interactions as they occur throughout a living cell would thus uniquely enable investigations of the intracellular landscape of [Ca] and its relationship to cell function. We have developed such a method based on capture of calmodulin-protein interactions by rapid photoactivated cross-linking (t ∼7s) in cells stably expressing a tandem affinity tagged calmodulin that have been metabolically labeled with a photoreactive methionine analog.

Evaluating Calmodulin-Protein Interactions by Rapid Photoactivated Cross-Linking in Live Cells Metabolically Labeled with Photo-Methionine.

This work addresses the question of how the Ca sensor protein calmodulin shapes cellular responses to Ca signals. Proteins interacting with affinity tagged calmodulin were captured by rapid ( ≈ 7 s) photoactivated cross-linking under basal conditions, after brief removal of extracellular Ca and during a cytosolic [Ca] transient in cells metabolically labeled with a photoreactive methionine analog. Tagged adducts were stringently enriched, and captured proteins were identified and quantified by LC-MS/MS.

Fluorescence quenching studies of structure and dynamics in calmodulin-eNOS complexes.

Activation of endothelial nitric oxide synthase (eNOS) by calmodulin (CaM) facilitates formation of a sequence of conformational states that is not well understood. Fluorescence decays of fluorescently labeled CaM bound to eNOS reveal four distinct conformational states and single-molecule fluorescence trajectories show multiple fluorescence states with transitions between states occurring on time scales of milliseconds to seconds. A model is proposed relating fluorescence quenching states to enzyme conformations.

Calmodulin-induced structural changes in endothelial nitric oxide synthase.

We have derived structures of intact calmodulin (CaM)-free and CaM-bound endothelial nitric oxide synthase (eNOS) by reconstruction from cryo-electron micrographs. The CaM-free reconstruction is well fitted by the oxygenase domain dimer, but the reductase domains are not visible, suggesting they are mobile and thus delocalized. Additional protein is visible in the CaM-bound reconstruction, concentrated in volumes near two basic patches on each oxygenase domain.

In calmodulin-IQ domain complexes, the Ca(2+)-free and Ca(2+)-bound forms of the calmodulin C-lobe direct the N-lobe to different binding sites.

We have investigated the roles played by the calmodulin (CaM) N- and C-lobes in establishing the conformations of CaM-IQ domain complexes in different Ca(2+)-free and Ca(2+)-bound states. Our results indicate a dominant role for the C-lobe in these complexes. When the C-lobe is Ca(2+)-free, it directs the N-lobe to a binding site within the IQ domain consensus sequence.