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PubMed1987

Preparation and properties of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

Peptides corresponding to the calmodulin-binding domain of MLCK and other target enzymes promise to be useful tools in the study of calmodulin action. Not only will they help to elucidate the molecular interactions of calmodulin with specific target enzymes, but they should also prove to be useful calmodulin antagonists because of their high affinity and high specificity. Given the lack of sequence similarity in the peptides presently known to avidly bind calmodulin, it seems as likely as not that there will be little sequence homology found between calmodulin-binding domains from different calmodulin-dependent enzymes. If this is the case, it may be difficult to identify the calmodulin-binding domain of a target enzyme by simple inspection of the enzyme's sequence. However, the techniques described in this chapter require only nominal amounts of target enzyme and should, therefore, prove useful in a situation where the sequence of a target enzyme is known from DNA cloning work, but only a small quantity of pure enzyme is available for structure-function studies.

Animals↗
PubMed1983

Calmodulin-dependent Ca2+-pump ATPase of human smooth muscle sarcolemma.

An enzymatically active Ca2+-stimulated ATPase has been isolated from the sarcolemmal sheets of human smooth muscle (myometrium). Ca2+-ATPase activity was quantitated in an assay medium which simulated the characteristic free ionic concentrations of the cytosol. New computer programs for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability constants, were used. In detergent-soluble form the enzyme has a high Ca2+-affinity expressed by an apparent Km (Ca2+) of 0.25 +/- 0.04 microM. The maximum specific activity (about 20 nmol of Pi/mg protein/min) was found in the micromolar domain of free-Ca2+ concentrations, the same levels required for normal maximal contractions in smooth muscle. The variation of free-Ca2+ concentration in the assay medium over 4 orders of magnitude (pCa 9 to pCa 5) resulted in a sigmoidal dependence of enzymatic activity, with a Hill coefficient of 1.4, which suggested the regulation of Ca2+-ATPase by allosteric effectors. The presence and the activator role of endogenous calmodulin in smooth muscle sarcolemma was proved by calmodulin-depletion experiments and by using suitable anticalmodulinic concentrations of trifluoperazine. The addition of exogenous calmodulin restored the enzyme activity. Apparently, the concentration of calmodulin in isolated smooth muscle sarcolemma is about 0.1% of sarcolemmal proteins, as deduced from the comparison of calmodulin-depletion and calmodulin-readdition experiments. Calmodulin increased significantly the enzyme Ca2+-affinity and Vmax (by a factor of about 10). At variance with the sarcoplasmic reticulum Ca2+-ATPase, the sarcolemmal Ca2+-ATPase is extremely sensitive to orthovanadate, half-maximal inhibition being observed at 0.8 microM vanadate. In conclusion, the Ca2+-ATPase isolated from smooth muscle sarcolemma appears very similar to the well-known Ca2+-pump ATPases of erythrocyte membrane, heart sarcolemma or axolemma. We suggest that this high-affinity Ca2+-ATPase represents the calmodulin-regulated Ca2+-extrusion pump of the smooth muscle sarcolemma.

Calcium↗
PubMed1987

Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase.

Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.

Animals↗
PubMed1989

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: divalent cation interactions monitored by direct binding and microcalorimetry.

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.

Animals↗
PubMed1990

Comparison of Ca(II), Cd(II), and Mg(II) titrations of tyrosine-99 spin-labeled bovine calmodulin.

Bovine calmodulin, spin-labeled at tyrosine-99, has been utilized in electron paramagnetic resonance (EPR) studies to investigate calmodulin interactions with Ca(II), Cd(II), and Mg(II). The addition of either Ca(II) or Cd(II) to apo-calmodulin results in a complex capable of activating target enzymes, such as 3', 5'-cyclic nucleotide phosphodiesterase (J. M. Buccigross, C. L. O'Donnell, and D. J. Nelson, Biochem. J. 235 677 [1986]), while Mg(II) is known to be incapable of activating calmodulin toward any of its target enzymes. Additions of Ca(II) and Cd(II) to spin-labeled apo-calmodulin gave rise to very similar changes in the EPR spectrum of the bound label, consistent with a dramatic decrease in the mobility of the nitroxide spin-label covalently attached to tyrosine-99. Addition of Mg(II) to spin-labeled apo-calmodulin caused no change in the EPR spectrum of the bound label. Thus, the conformational changes induced by Ca(II) and Cd(II) ion binding to calmodulin, which lead to decreased tyrosine-99 spin label mobility, are clearly not occurring when Mg(II) ion binds. These results are consistent with the results of other spectroscopic studies, which indicate that "activating" metal ions, such as Ca(II) and Cd(II), produce calmodulin conformers that are different from those produced by "inactivating" metal ions, such as Mg(II).

Animals↗
PubMed1983

Aluminum interaction with calmodulin. Evidence for altered structure and function from optical and enzymatic studies.

The interaction of aluminum ions with bovine brain calmodulin has been examined by fluorescence spectroscopy, circular dichroic spectrophotometry and equilibrium dialysis, and by the calmodulin-dependent activation of 3',5'-cyclic nucleotide phosphodiesterase. These experiments show that aluminum binds stoichiometrically and cooperatively to calmodulin. Binding of aluminum at a molar ratio of 2:1 to calmodulin suffices to induce a major structural change. Estimates from spectroscopic data indicate that the binding affinity for the first mol of aluminum bound to the protein is about one order of magnitude stronger than that of calcium to its comparable site. These estimates agree with a dissociation constant of 0.4 microM derived from equilibrium dialysis experiments. Interaction of aluminum with calmodulin induces a helix-coil transition and enhances the hydrophobic surface area much more than calcium does. A molar ratio of 4:1 for [aluminum]/[calmodulin] is sufficient to block completely the activity of the calcium-calmodulin-dependent phosphodiesterase. Highly hydrated aluminum ions apparently promote solvent-rich, disordered polypeptide regions in calmodulin which, in turn, profoundly influence the protein's flexibility.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Characterisation of calmodulin from Drosophila heads.

Calmodulin from Drosophila heads has been purified to apparent electrophoretic homogeneity. It has the same characteristics as bovine brain calmodulin with respect to the migration upon polyacrylamide gel electrophoresis and maximal activation of a calmodulin-deficient cAMP phosphodiesterase. The amino acid composition resembles bovine brain calmodulin with the exception that trimethyllysine is absent and that it contains only one tyrosine. The tryptic peptide map of Drosophila calmodulin suggests some differences in the amino acid sequence as compared to bovine brain calmodulin. These proposed differences in the primary structure may explain why Drosophila calmodulin is less potent than bovine brain calmodulin in the activation of a cAMP phosphodiesterase from bovine brain.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

A calmodulin endoproteinase from mitochondrial membranes.

A proteinase specific for calmodulin has been identified in a crude rat kidney Triton-extracted or sonicated mitochondrial fraction and solubilized by EGTA extraction of these membranes. Mitochondrial fractions from other tissues had less activity, with relative activities: kidney = spleen greater than testes greater than liver, and no detectable activity in either brain or skeletal muscle. This enzyme is active in the presence of EGTA, but not in the presence of calcium, and cleaves calmodulin into three major peptide fragments with Mr 6000, 9000 and 10,000. N-methylated and non-methylated calmodulins were both cleaved by calmodulin proteinase and while troponin was a poor substrate, it was cleaved in the presence of either calcium or EGTA. No other EF hand calcium-binding proteins or other major mitochondrial proteins were cleaved by this enzyme. The peptides resulting from calmodulin proteinase action were isolated by reverse-phase high performance liquid chromatography (HPLC) and sequenced. Sequence analysis indicated that calmodulin proteinase cleaves calmodulin at Lys-75. The effects of proteinase inhibitors indicate that calmodulin proteinase is a trypsin-like enzyme belonging to the serine endopeptidase family of enzymes.

Amino Acid Sequence↗
PubMed1983

The effect of calmodulin on histamine release in the rat peritoneal mast cell.

To investigate the role of the Ca2+-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound 48/80, polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca2+. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release.

Animals↗
PubMed1989

Calmodulin binding to the intestinal brush-border membrane: comparison to other calcium-binding proteins.

The intestinal brush-border membrane contains a high concentration of calmodulin bound to a 105,000 dalton (105 kDa) protein. Binding of radioiodinated calmodulin to this protein does not require calcium but is inhibited by trifluoperazine and excess unlabelled calmodulin. Recent evidence suggests that the 105 kDa protein in conjunction with calmodulin may be involved in the regulation of calcium transport across the brush-border membrane. In this report, we evaluated the binding of the 105 kDa protein to other radioiodinated calcium-binding proteins including the vitamin D-dependent intestinal calcium-binding protein. We observed that troponin C and S100 beta protein both bound strongly to the 105 kDa protein. The binding of S100 beta was inhibited by EGTA, but was little affected by trifluoperazine and excess unlabelled S100 beta, whereas that of troponin C was inhibited by trifluoperazine and excess unlabelled troponin C, but was little affected by EGTA. Both troponin C and S100 beta bound to a large number of proteins to which calmodulin did not bind. The vitamin D-dependent calcium-binding protein (calbindin) from chick intestine and rat kidney also bound to the 105 kDa protein, albeit more weakly than troponin C, S100 beta and calmodulin. The binding of the calbindins was increased by EGTA and was little affected by trifluoperazine and excess unlabelled calbindin. Parvalbumin, rat osteocalcin, and alpha-lactalbumin showed little binding to any brush-border membrane protein. Our results indicate that the 105 kDa calmodulin-binding protein of the intestinal brush border can bind to a variety of calcium-binding proteins all of which contain homologous regions thought to be the calcium-binding sites. Only the binding of troponin C resembles the binding of calmodulin, however, in being inhibited by trifluoperazine and excess unlabelled ligand. The functional significance of these observations in terms of regulating calcium transport across the brush-border membrane remains to be established.

Animals↗
PubMed1983

Calmodulin stimulates polyamine-responsive protein kinase in the absence of Ca2+.

A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540-3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of calmodulin did not require Ca2+ for activation of the enzyme; and activation occurred in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca2+. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased Vmax and with no changes in the Km (ATP). High levels of cation, up to 100 mM Mg2+, did not effect the action of calmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue.

Animals↗
PubMed1991

Multiple analysis of tyrosine hydroxylase and calmodulin distributions in the forebrain of the rat using a microphotometry system.

Immunohistochemical distributions of tyrosine hydroxylase and calmodulin in the rat forebrain were analyzed quantitatively to confirm our previous results that the activities of central catecholamine-synthesizing enzymes are regulated by a calcium-calmodulin-dependent system. The adjacent slices of adult rat brain were stained immunohistochemically for tyrosine hydroxylase and for calmodulin, and the distributions and amounts of these proteins were measured by a fluorescence microphotometry system that was developed in our laboratory. Immunohistochemical fluorescence intensity was measured stepwise at 40 microns intervals through a 6 microns phi (on the slice) pin hole. Each stained brain slice was divided into approximately 100,000 areas, and measured for fluorescence intensity and displayed two- and three-dimensionally. Immunoreactive staining of tyrosine hydroxylase and calmodulin was observed in almost all areas of the brain, but its intensity varied. The relatively high levels of calmodulin could be observed in brain regions with high levels of tyrosine hydroxylase distribution, though high levels of tyrosine hydroxylase could not always be observed in brain regions where high levels of calmodulin were distributed. In the present study, high levels of tyrosine hydroxylase and calmodulin were distributed in the nucleus accumbens septi and the lateral part of the neostriatum regions in which the amount of dopamine was increased by the intraventricular administration of calcium. These findings suggest that the synthesis of central catecholamines is regulated by a calcium-calmodulin-dependent system.

Animals↗
PubMed1990

GAP-43 as a 'calmodulin sponge' and some implications for calcium signalling in axon terminals.

In most neurons, the maturation of axonal growth cones to become stable synaptic terminals is accompanied by a dramatic decline in the abundance of a major growth cone component, GAP-43. Accumulation of GAP-43 persists, however, in a minority of mature synaptic terminals. What properties of axons and their terminals are affected by these changes in GAP-43 expression? Storm and colleagues first noted that the membrane- and calmodulin-binding properties of GAP-43 (a.k.a. P-57 or neuromodulin) could allow it to sequester a large fraction of calmodulin to the submembranous regions, and to release free calmodulin in response to protein kinase C activation. Analysis of evolutionarily conserved sequences in GAP-43 indicates that these properties are central to the biological effects of the protein. If GAP-43 is presumed to inactivate bound calmodulin, the network of GAP-43 in an axon terminal could be considered a regulatable calmodulin buffer, or 'calmodulin sponge', absorbing free calmodulin and releasing it in response to activation of protein kinase C. Such a calmodulin sponge has properties that could be useful in modulating the responses of membrane and cytoskeletal assembly events to calcium signals in growth cones, and in mediating long-term potentiation of neurotransmitter release from some pre-synaptic terminals.

Animals↗
PubMed1995

Target-peptide-induced changes in the structure and dynamics of calmodulin as probed by frequency domain fluorimetry of bound Tb(III).

Tb(III) luminescence is used to probe the conformational change induced in the calcium regulatory protein calmodulin upon binding to a target peptide. The luminescence lifetime for Tb(III) measured by frequency domain fluorimetry increases from 1278 microseconds for the calmodulin complex to 1496 microsecond for the complex of calmodulin and M13, a peptide derived from the calmodulin target protein myosin light chain kinase. The intensity of the Tb(III) emission increases over the solution value by a factor of 726 and 891 when bound to calmodulin and to the complex of calmodulin and M13 respectively. The sensitivity of the Tb(III) decay rate to deuterated solvent was also measured and is consistent with a single water molecule bound to the metal in both the calmodulin and calmodulin-M13 complex. The most dramatic change induced by M13 is the threefold reduction in the width of the Tb(III) lifetime distribution, which is interpreted to be a target-peptide-induced annealing of the previously flexible metal-binding site.

Amino Acid Sequence↗
PubMed2006

Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme.

A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the L-citrulline formation of nNOS from L-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.

Allosteric Regulation↗
PubMed2004

Calcium negatively modulates calmodulin interaction with IQGAP1.

IQGAP1 regulates cytoskeletal dynamics through interactions with the Rho family GTPases Rac1 and Cdc42, F-actin, and beta-catenin. Calmodulin interaction with IQ motifs of IQGAP1 negatively influences these IQGAP1 interactions. Although, calmodulin interacts with IQGAP1 in the absence of Ca(2+) and was suggested to exhibit reduced binding when Ca(2+) bound, recent reports show substantially greater binding when Ca(2+) is present. Binding evaluations have primarily relied on IQGAP1 interaction with calmodulin conjugated to Sepharose 4B. In this study we evaluated the Ca(2+)-dependence of calmodulin interaction with native IQGAP1 using a series of independent biochemical approaches. We found the apparent binding of calmodulin to IQGAP1 was Ca(2+)-independent, being between 5- and 20-fold greater in the absence than in the presence of Ca(2+). In addition, calmodulin interaction with IQGAP1 was negatively regulated by buffer [Ca(2+)] (IC(50)=3.4x10(-7)M). Regulation was specific to Ca(2+), as Ba(2+) was approximately 400-fold less effective than Ca(2+) at modulating the interaction. Moreover, testing of calmodulin mutants demonstrated that apocalmodulin tightly binds IQGAP1 and that the N- and C-terminal pair of EF hands are important for Ca(2+) sensitivity. These data indicate that calmodulin may disassemble from IQGAP1 to facilitate IQGAP1 interaction with effectors of cytoskeletal reorganization during conditions of cell activation that promote increased cytosolic [Ca(2+)].

Animals↗
PubMed2004

The association of calmodulin with central spindle regulates the initiation of cytokinesis in HeLa cells.

Calmodulin is a major cytoplasmic calcium receptor that performs multiple functions in the cell including cytokinesis. Central spindle appears between separating chromatin masses after metaphase-anaphase transition. The interaction of microtubules from central spindle with cell cortex regulates the cleavage furrow formation. In this paper, we use green fluorescence protein (GFP)-tagged calmodulin as a living cell probe to examine the detailed dynamic redistribution and co-localization of calmodulin with central spindle during cytokinesis and the function of this distribution pattern in a tripolar HeLa cell model. We found that calmodulin is associated with spindle microtubules during mitosis and begins to aggregate with central spindle after anaphase initiation. The absence of either central spindle or central spindle-distributed calmodulin is correlated with the defect in the formation of cleavage furrow, where contractile ring-distributed CaM is also extinct. Further analysis found that both the assembly of central spindle and the formation of cleavage furrow are affected by the W7 treatment. The microtubule density of central spindle was decreased after the treatment. Only less than 10% of the synchronized cells enter cytokinesis when treated with 25 microM W7, and the completion time of furrow regression is also delayed from 10 min to at least 40 min. It is suggested that calmodulin plays a significant role in cytokinesis including furrow formation and regression, The understanding of the interaction between calmodulin and microtubules may give us insight into the mechanism through which calmodulin regulates cytokinesis.

Anaphase↗
PubMed2005

Calorimetry and mass spectrometry study of oxidized calmodulin interaction with target and differential repair by methionine sulfoxide reductases.

Calmodulin is known to be a target for oxidation, which leads to conversion of methionine residues to methionine sulfoxides. Previously, we reported that both methionine sulfoxide reductases MsrA and MsrB were able to reduce methionine sulfoxide residues in oxidized calmodulin. In the present study, we have made use of the interaction between calmodulin and RS20, a peptide model for calmodulin targets, to probe the structural consequences of oxidation and mode of repair both by MsrA and MsrB. Isothermal titration calorimetry and differential scanning calorimetry showed that oxidized calmodulin interacts with RS20 via its C-terminal domain only, resulting in a non-productive complex. As shown by spectrofluorometry, oxidized calmodulin treated with MsrA exhibited native binding affinity for RS20. In contrast, MsrB-treatment of oxidized calmodulin resulted in 10-fold reduced affinity. Mass spectrometry revealed that the sulfoxide derivative of methionine residue 124 was differentially repaired by MsrA and MsrB. This provided a basis for rationalizing the difference in binding affinities of oxidized calmodulin reported above, since Met124 residue had been shown to be critical for interaction with some targets. This study provides the first evidence that in an oxidized polypeptide chain MetSO residues might be differentially repaired by the two Msr enzymes.

Animals↗