Methionine oxidation in calmodulin (CaM) isolated from senescent brain results in an inability to fully activate the plasma membrane (PM) Ca-ATPase, which may contribute to observed increases in cytosolic calcium levels under conditions of oxidative stress and biological aging. To identify the functional importance of the oxidation of Met(144) and Met(145) near the carboxyl-terminus of CaM, we have used site-directed mutagenesis to substitute leucines for methionines at other positions in CaM, permitting the site-specific oxidation of Met(144) and Met(145). Prior to their oxidation, the CaM-dependent activation of the PM-Ca-ATPase by these CaM mutants is similar to that of wild-type CaM. Likewise, oxidation of individual methionines has a minimal effect on the CaM concentration necessary for half-maximal activation of the PM-Ca-ATPase. These results are consistent with previous suggestions that no single methionine within CaM is essential for activation of the PM-Ca-ATPase. Oxidation of either Met(144) and Met(145) or all nine methionines in CaM results in an equivalent inhibition of the PM-Ca-ATPase, resulting in a 50-60% reduction in the level of enzyme activation. Oxidation of Met(144) is largely responsible for the decreased extent of enzyme activation, suggesting that this site is critical in modulating the sensitivity of CaM to oxidant-induced loss-of-function. These results are discussed in terms of a possible functional role for Met(144) and Met(145) in CaM as redox sensors that function to modulate calcium homeostasis and energy metabolism in response to conditions of oxidative stress.

Research on the one-electron reduced analogue of NO, namely nitroxyl (HNO/NO–), has revealed distinguishing properties regarding its utility as a therapeutic. However, the fleeting nature of HNO requires the design of donor molecules. Metal nitrosyl (MNO) complexes could serve as potential HNO donors. The synthesis, spectroscopic/structural characterization, and HNO donor properties of a {CoNO}8 complex in a pyrrole/imine ligand frame are reported. The {CoNO}8 complex [Co(LN4PhCl)(NO)] (1) does not react with established HNO targets such as FeIII hemes or Ph3P. However, in the presence of stoichiometric H+1 behaves as an HNO donor. Complex 1 readily reacts with [Fe(TPP)Cl] or Ph3P to afford the {FeNO}7 porphyrin or Ph3P=O/Ph3P=NH, respectively. In the absence of an HNO target, the {Co(NO)2}10 dinitrosyl (3) is the end product. Complex 1 also reacts with O2 to yield the corresponding CoIII-η1-ONO2 (2) nitrato analogue. This report is the first to suggest an HNO donor role for {CoNO}8 with biotargets such as FeIII-porphyrins.

The solution structure of a de novo designed disulfide-bridged two-α-helix peptide that self-assembles to form a 2-fold symmetric four-α-helix bundle protein (α‘-SS-α‘)2 has been solved by NMR spectroscopy. The 33-residue peptide, (α‘-SH), that is the basic building block of the bundle has been recombinantly expressed. The three-dimensional structure of the asymmetric unit of the bundle has been determined using interproton distance restraints derived from the nuclear Overhauser effect (NOE), covalent torsion angle restraints derived from three bond scalar coupling constants, and longer range angular restraints derived from residual dipolar couplings. The covalent α‘-SS-α‘ unit forms a pair of parallel α-helices that use heptad a-, d-, e-, and g-side chains to form a hydrophobic core extending the length of the molecule. The distribution of polar and nonpolar side chains on the surface of α‘-SS-α‘ structure is asymmetric. The hydrophilic face is comprised of glutamate and lysine side chains, while the opposite face is comprised of leucine, isoleucine, phenylalanine, tryptophan, and neutral histidine side chains. Equilibrium sedimentation analysis, size-exclusion chromatography, pulsed field gradient translation diffusion measurements, and a rotational correlation time derived from 15N NMR relaxation studies all indicate that the covalent α‘-SS-α‘ unit forms a noncovalent dimer, (α‘-SS-α‘)2, in solution. The structure confirms many expected design features and illuminates an apparent dichotomy of structure where the helical interface of the disulfide bridged two-α-helix peptide appears nativelike while the adjacent, noncovalent interface shows non-nativelike behavior. Available evidence indicates the four α-helix bundle can adopt either an anti or syn topology. The structure is discussed with respect to the potential origins of conformational specificity and nativelike protein structure.

A calmodulin (CaM) mutant (T34,110C-CaM) doubly labeled with fluorescence probes AlexaFluor 488 and Texas Red in opposing domains (CaM-DA) has been used to examine conformational heterogeneity in CaM by single-pair fluorescence resonance energy transfer (spFRET). Burst-integrated FRET efficiencies of freely diffusing CaM-DA single molecules yielded distributions of distance between domains of CaM-DA. We recently reported distinct conformational substates of Ca(2+)-CaM-DA and apoCaM-DA, with peaks in the distance distributions centered at approximately 28 A, 34-38 A, and 55 A [Slaughter et al. (2004) J. Phys. Chem. B 108, 10388-10397]. In the present study, shifts in the amplitudes and center distances of the conformational substates were detected with variation in solution conditions. The amplitude of an extended conformation was observed to change as a function of Ca(2+) over a free Ca(2+) range that is consistent with binding to the high affinity, C-terminal Ca(2+) binding sites, suggesting the existence of communication between lobes of CaM. Lowering pH shifted the relative amplitudes of the conformations, with a marked increase in the presence of the compact conformations and an almost complete absence of the extended conformation. In addition, the single-molecule distance distribution of apoCaM-DA at reduced ionic strength was shifted to longer distance and showed evidence of an increase in conformational heterogeneity relative to apoCaM-DA at physiological ionic strength. Oxidation of methionine residues in CaM-DA produced a substantial increase in the amplitude of the extended conformation relative to the more compact conformation. The results are considered in light of a hypothesis that suggests that electrostatic interactions between charged amino acid side chains play an important role in determining the most stable CaM conformation under varying solution conditions.