Properties Of Calmodulin Research Articles

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

Calmodulin plays a vital role in mediating bidirectional synaptic plasticity by activating either calcium/calmodulin-dependent protein kinase II (CaMKII) or protein phosphatase 2B (PP2B) at different calcium concentrations. We propose an allosteric model for calmodulin activation, in which binding to calcium facilitates the transition between a low-affinity [tense (T)] and a high-affinity [relaxed (R)] state. The four calcium-binding sites are assumed to be nonidentical. The model is consistent with previously reported experimental data for calcium binding to calmodulin. It also accounts for known properties of calmodulin that have been difficult to model so far, including the activity of nonsaturated forms of calmodulin (we predict the existence of open conformations in the absence of calcium), an increase in calcium affinity once calmodulin is bound to a target, and the differential activation of CaMKII and PP2B depending on calcium concentration.

Free-energy simulations of the oxidation of c-terminal methionines in calmodulin.

In the course of aging or under conditions of oxidative stress, methionine residues of calmodulin undergo oxidation, leading to loss of biological activity of the protein. We have performed free-energy simulations of the effects of C-terminal methionine side-chain oxidation on the properties of calmodulin. The simulation results indicate that oxidation should have a destabilizing effect on all three protein functional states: calcium free, calcium loaded, and with both calcium and target peptide bound. Because the different states are destabilized by different amounts, this leads to a more complex pattern in the observable effects on protein thermal stability, calcium affinity, and binding of a target peptide. The influence of oxidation on the free energy of CaM unfolding is estimated by comparing the free-energy cost of oxidizing a Met residue in a Gly-Met-Gly peptide and in the protein. The protein thermal stability of the oxidized forms is lowered by a moderate amount 1-3 kcal/mol, in qualitative agreement with experimental results of 0.3 kcal/mol. The calculated changes in affinity for calcium and for the target peptide show opposing trends. Oxidation at position 144 is predicted to enhance peptide binding and weaken calcium binding, whereas oxidation at 145 weakens peptide binding and enhances affinity for calcium. The lower affinity of Met 145-oxidized calmodulin toward the target peptide correlates with experimentally observed lowering of calmodulin-activated Ca-ATPase activity when oxidized calmodulin from aged rat brains is used. Thus, our simulations suggest that Met 145 is the oxidation site in the C-terminal fragment of calmodulin. The microscopic mechanism behind the calculated free energy changes appears to be a greater affinity for water of the oxidized Met side-chain relative to normal Met. Structures with Met exposed to solvent had consistently lower free energies than those with buried Met sidechains.

Organization of the Drosophila genome in mutants with changes in second messenger metabolism and learning ability.

Mechanisms modifying the structural-functional organization of polytene chromosomes were studied in a Drosophila line in which the activating properties of calmodulin were altered and learning ability was increased, by treating mutants with homeopathic preparations which affect Ca2+ and F- ion metabolism. The results indicated a dominant role for Ca2+ ions and calmodulin in determining the chromocentric organization of the nucleus. F- ions, which stimulate the adenylate cyclase complex, were found not to have a role.

Isolation and characterization of calmodulin and a novel calcium-binding protein calpollenin from Pinus yunnanensis pollen

A protein identifiable as calmodulin (CaM) was separated from Pinus yunnanensis pollen by phenyl-sepharose affinity chromatography and further purified by preparative polyacrylamide gel electrophoresis (PAGE). This pollen CaM is heat stable, could activate cAMP phosphodiesterase in a Ca 2+-dependent fashion, and showed the migration change on sodium dodecyl sulfate (SDS)-PAGE in the presence or absence of Ca 2+. Its UV absorption spectrum showed five characteristics peaks. One third of its amino acid composition was acidic amino acids, and the Phe/Tyr ratio was 8:2. In general, the properties of CaM from Pinus yunnanensis pollen is similar to CaMs from animal and other plant sources. A novel 21-kDa calcium-binding protein (CaBP), given the name of calpollenin, was isolated and purified from P. yunnanensis pollen by following the same procedure as for CaM extraction. It was extracted more easily by Triton-containing buffer, and precipitated reversibly by 3.0% (w/v) trichloroacetic acid. Similar to CaM, this calpollenin could bind to a hydrophobic ligand in a Ca 2+-dependent manner, and showed an altered electrophoretic mobility on SDS-PAGE in the presence or absence of Ca 2+. However, calpollenin was heat unstable, and was denatured on heating at 90°C for 5 min. Its UV absorption spectrum showed a single peak at 275 nm. Analysis of amino acid composition demonstrated that calpollenin contained approximately 213 amino acid residues, among which there was a higher proportion of acidic (24.8%) compared with basic amino acids (5.2%).

Calmodulin-binding proteins of calcium-independent type in rat brain synaptosomal membranes: Their localization and properties

Some properties of calmodulin (CaM)-binding proteins (CaMBPs) of the Ca 2+-independent type were investigated in the synaptosomal membrane (SM) from rat brain using the [ 125I]CaM gel overlay method. When SM was prepared in the presence of Ca 2+, Ca 2+-independent CaM binding was decreased, whereas the Ca 2+-dependent type was not altered. All Ca 2+-independent-type CaMBPs were membrane-bound and scarcely present in the soluble fractions. When SM was heat-denatured, the 24 22.5 -kDa CaMBPs could no longer be detected by [ 125]CaM binding and a new component with higher molecular mass (>200kDa) was shown to bind CaM in a Ca 2+-independent manner. A possible effect of cAMP- and Ca 2+ CaM -dependent phosphorylation on CaM binding was also examined.

Exposure of hydrophobic sites in CA2+‐binding proteins: Influence on chromatographic properties and structure‐function relationships

AbstractCalmodulin and related Ca2+ ‐binding proteins were characterized using different labeling, chromatographic and spectroscopic techniques. Ca2+‐binding to members of the so‐called “EF‐hand”‐ or “helix‐loop‐helix”‐model protein family induced conformational changes, thereby exposing hydrophobic sites in some of them. These sites were identified using the photoreactive, carbene‐generating, radioactive probe 3‐(trifluoromethyl)‐3‐(m‐[1251]iodophenyl)diazirine, and characterized using different high performance liquid chromatography techniques. The influence of chemical modification of some of the amino acid residues on the properties of calmodulin were characterized using circular dichroism. A correlation between an increase of oxidized methionine residues of calmodulin and a decrease of Ca2+‐induced helical content was observed.

Vitamin D-3 induces the de novo synthesis of calmodulin in Phaseolus vulgaris root segments growing in vitro

Vitamin D-3 affects growth and Ca 2+ transport in Phaseolus vulgaris roots by a mechanism dependent on de novo protein synthesis. The objective of this work was to identify the protein(s) induced by the sterol. Phaseolus vulgaris root segments cultured in vitro were used. Protein extracts of control cultures and cultures treated with 10 −9 M vitamin D-3 for 2 h labelled with [ 14C]leucine and [ 3H]leucine, respectively, were mixed and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Examination of 3H: 14C ratios in slices of gels revealed that vitamin D-3 stimulates production of a protein which exhibited Ca 2+-dependent electrophoretic mobility. The apparent molecular weight was 14500 in the presence of 1 mM Ca 2+ and 18 000 in the presence of 5 mM EGTA. This protein was heat- and acid-stable, bound 45Ca 2+ and had an isoelectric pH of 3.8-4.1. These data are consistent with the properties of calmodulin. In agreement with these observations, higher levels of calmodulin were detected in roots treated with vitamin D-3 than in control roots by means of assays based on the activation of calmodulin-dependent enzymes. Moreover, estimation of calmodulin relative synthesis and degradation rates in roots cultures in the absence and presence of vitamin D-3 indicated that the sterol increases calmodulin synthesis without affecting its degradation. In addition, the results suggest that the synthesis of other low-molecular-weight Ca 2+-binding proteins may be affected by vitamin D-3.

Calcium-binding and its effect on circular dichroism of plant calmodulin

The Ca(2+)-binding properties of calmodulin purified from zucchini (Cucurbita pepo L.) has been determined. A value of 3.3 mol Ca(2+) per mol of zucchini calmodulin was measured at pH 7.5 by equilibrium chromatography. The far-and near-UV circular-dichroic spectra of the Ca(2+)-and Mg(2+)-saturated as well as from the metal-free forms of zucchini calmodulin reveal that upon Ca(2+)-binding the α-helix content increases. A comparison with the spectra of vertebrate calmodulin indicates that both calmodulin have a similar secondary structure, similar Ca(2+)-induced conformational changes and the same number of Ca(2+)-binding sites.

Effects of cation binding on the thermal transitions in calmodulin

The thermal transitions in different forms of bovine brain calmodulin (0, 1, 2, 3 and 4 bound Ca 2+ ions per molecule) have been studied by means of microcalorimetry, intrinsic tyrosine fluorescence, circular dichroism and infrared spectroscopy. The heating of the apoprotein from 5 to 110°C induces at least three unfolding transitions. The heating of Ca 2+-loaded calmodulin causes at least two structural transitions, one of which occurs at relatively low temperatures, from approx. 30 to approx 50°C. The binding of the biologically significant Ca 2+, Mg 2+, Na + and K + ions has been measured at 12, 20, 28, 37 and 50°C by means of the fluorescence method. The values of the binding parameters for these cations do not depend on temperature within the range 12 to 50°C. It has been proposed that the temperature independence of the metal-ion-binding properties of calmodulin is achieved due to the temperature-induced structural changes, which adjust the protein conformation in such a way that the protein-binding parameters remain constant.

Purification and properties of calmodulin from adrenal cortex

Calmodulin (CaM), a multifunctional calcium binding protein with no known enzymatic activity, has been purified to homogeneity from bovine adrenal cortex. The purification included anion exchange on DE-52 cellulose, ammonium sulfate precipitation, and separation by molecular sieving on Sephadex G-150. The yield of CaM from 900 g of whole adrenal was 150 mg. Adrenocortical CaM showed a molecular weight of 18,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, an isoelectric point of 4.1, and demonstrated a characteristic shift in mobility on polyacrylamide gels in the presence of calcium. The spectral properties of adrenocortical CaM differed slightly from those of CaM isolated from bovine brain. Minor differences were observed in peptide maps and amino acid composition between adrenocortical and brain CaM, but adrenocortical CaM contained a single trimethyllysine residue characteristic of all mammalian forms of CaM isolated to date. Adrenocortical CaM is biologically active in the stimulation of activator-deficient phosphodiesterase, and showed a half-maximal effective concentration (EC 50) of 3 n m for stimulation of adenylate cyclase from Bordetella pertussis.

Characterization and purification of a soluble protein controlling Ca-channel activity in paramecium

The analysis of the voltage-sensitive Ca ++ channel of the unicellular eucaryote, Paramecium has been extended to a biochemical level based on recent observations that the transfer of cytoplasm from wild-type cells into mutants lacking Ca ++-channel function (“pawn” in P. tetraurelia and “CNR” in P. caudatum) causes mutant cells to regain Ca ++-channel function. We have microinjected various cytoplasmic fractions into mutant cells and measured the restored Ca ++-channel function using a convenient behavioral assay. Following the “curing” activity, we characterized and purified the component from wild-type cytoplasm that can restore the function missing in cells carrying mutations in the cnrC gene. The curing factor is not an RNA, but a heat-labile, -SH-containing protein that appears to affect existing mutant channels on the ciliary membrane. We have purified this factor over 500-fold from the soluble cytoplasm using conventional techniques. The protein is of low apparent molecular weight (<30,000 daltons), acidic, soluble, and does not have the properties of calmodulin.