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Dynamics of Nitric Oxide Synthase Calmodulin Interactions at Physiological Calcium Concentrations.

In this manuscript we show that the CaM-NOS complexes have similar structures at physiological and fully saturated Ca levels, however, their dynamics are remarkably different. At 225 nM Ca-concentrations the CaM-NOS complexes show overall an increase in backbone dynamics, when compared to the dynamics of the complexes under saturating Ca concentrations. Specifically, the N-lobe of CaM in the CaM-iNOS complex displays lower internal mobility and higher exchange protection compared to that of the CaM-eNOS complex. In contrast, the C-lobe of CaM in the CaM-eNOS complex is less dynamic. These results illustrate that structures of CaM-NOS complexes determined at saturated Ca-concentrations cannot provide a complete picture because the differences in intramolecular dynamics only become visible at physiological Ca levels. (Piazza et al., 2015)

Chemical Shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides. 

In this paper, we present backbone and sidechain NMR resonance assignments of modified CaM interacting with NOS peptides. This work provides the basis for a detailed study of CaM-NOS interaction dynamics using nitrogen-15 relaxation methods. (Piazza et al., 2015)

Solution Structure of Calmodulin bound to the target peptide of Endothelial Nitric Oxide Synthase phosphorylated at Thr495. 

We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size eNOS in order to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an alpha-helix. The calcium affinity of the CaM-target peptide complex is reduced due to phosphorylation and this leads to weaker binding under low physiological calcium concentrations. This study provides an explanation for the reduced NO production by eNOS carrying a phosphorylated Thr495 residue. (Piazza et al., 2014)

Pre-bound ligands can alter the binding properties of higher affinity ligands.

We have used competitive binding studies using isothermal titration calorimetry and stopped flow kinetics to explore the adaptive nature of RNA–ligand interactions. The results of these studies reveal that binding of one ligand can reduce the ability of the aptamer pocket to adapt to another ligand, even if this second ligand has a significantly higher affinity to the free aptamer. (Da Costa et al., 2013)

The effect of a phosphomimetic mutation on the interaction between calmodulin and nitric oxide synthase.

Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. Our structure and biophysical studies illustrate how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes. (Piazza et al., 2012)

Entropy and magnesiuum ions control the specificity of ligand binding in an a RNA aptramer.

We have used isothermal titration calorimetry to show that the malachite green binding RNA aptamer switches its preference for binding from the original ligand malachite green to tetramethylrosamine in prersence of high salt concentrations or divalent metal ions. (Da Costa et al., 2011)