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Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophilamelanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species., Introduction [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase with important roles in cardiovascular physiology and disease (1-4). Despite the critical role of CaMKII in fight-or-flight responses and human [...]

Research has been conducted on the use of the C-terminal tail in stabilizing the EF-hand protein. Results indicate that the solvent-exposed regions can affect the protein stability.

Research has been conducted on the site-specific substitution of Tyr (super)138 with Phe (super)138 or Gln (super)138. The effect of this substitution on the structural coupling between the opposing calmodulin domains has been investigated via the circular dichroism and frequency-domain fluorescence spectroscopy.

Results demonstrate that in the wild-type Paramecium melittin binds to the C-domain of the calmodulin in the absence and/or presence of calcium as revealed by analysis of the binding activity in the mutants. Data further show tertiary collapse of calmodulin upon calcium binding to the N-domain of the protein.

Research has been conducted on the eykaryotic protein calmodulin. Results indicate that domain-specific mutants of Paramecium calmodulin affect the ion channels differentially.

The two domains of calmodulin have unique properties and may have different mechanisms in activating targets. The domains are cloned and the calcium binding affinities and the stability of the domains were determined independently. Results reveal that the amino terminal domain binds calcium more avidly and is protected from proteolysis. The isolated amino terminal domain also exhibits higher binding affinity compared to the amino terminal domain of native calmodulin. The implications of these findings are discussed.

Calmodulin acts as an intracellular calcium receptor in calcium-induced switching in eukaryotic cells. Calmodulin's ligation states were examined using thrombin footprinting techniques, with each ligand state analysed for energetic and structural properties. Calcium saturation protected the Arg37 and Arg106 sites from proteolysis. The susceptibility/protection of ligands was dependent on the level of calcium and the sites to which the calcium binds.

The calcium dependent structural changes of calmodulin are studied by nuclear magnetic resonance spectroscopy (NMR) by an equilibrium titration, and the isotherms of the individual site binding are determined. The concentration of macromolecule needed for required signal intensity is larger compared to the dissociation constant so the equilibrium titration of high affinity ligand binding cannot be obtained by NMR. A study of discontinuous equilibrium titration permits the investigation of stoicism binding properties.

A new proteolytic footprinting technique uses endoproteinase GluC (EndoGluC) to analyze the residue-specific conformational changes in calmodulin due to binding by calcium. Individual peptide bonds are significantly modified upon calcium binding. Quantitative EndoGluC susceptibility profiles for E31 and E87 indicate that calmodulin adopts three distinct conformations. N-terminal domain exhibits significant interaction with C-terminal domain. Equilibrium constants of the binding suggest cooperative binding within each domains.

The study of 10 ligation species of CN-methemoglobin (CN-met) in the alkaline Bohr effect region reveals the free energies of quaternary assembly of dimers to tetramers. The CN-met species reveals two different values of Bohr free energy and Bohr proton release at each pH value. Symmetry rule influences proton release and free energy.

Circular dichroism, fluorescein emission and fluorescence spectroscopy are used for studying the interaction between calmodium (CaM) and [Na.sub.V]1.2 (voltage dependent sodium channel type II) at varying calcium concentrations. The studies have shown that IQ motif has led to a selective decrease in the calcium affinity of the C-domain to an extent such that the N-domain has a slightly higher affinity than the C-domain.

The complex interplay between [Ca.sup.2+}, calmodulin (CaM), calmodulin-dependent kinase II (CaMKII) and [alpha]-actinin for binding to the N-methyl-D-aspartate (NMDA) receptor NR1 C0 region is analyzed. The results have shown an intricate mechanism where [Ca.sup.2+] has functioned to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII.

Calcium-binding energetic of 11 viable mutants of PCaM identified by their effects on ion-channel regulation and ensuing irregular stimulus-avoidance behavior of Paramecia were studied by observing calcium-dependent decreases in intrinsic Phe fluorescence that indicated the occupancy of sites I and II in the N-domain and increases in intrinsic Tyr IV in the C-domain. The results suggest that while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel.

Human voltage-gated sodium (NaV) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel. [ABSTRACT FROM AUTHOR]