Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum

Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of either erythrocyte membrane Ca2+-ATPase activity or cardiac sarcoplasmic reticulum phosphorylation are very similar. Affinity labeling of troponin subunits with 125I- and benzophenone-4-maleimide-labeled CaM demonstrates CaM binding to troponin I (TnI) and troponin T (TnT) in binary complexes, as well as to both subunits in the CaM.TnI.TnT ternary complex. This suggests that both subunits are within 10 A of Cys27 of calmodulin. Affinity labeling of cardiac sarcoplasmic reticulum vesicles with 125I- and benzophenone-4-maleimide-labeled CaM exhibits a Ca2+- and Mg2+-dependent labeling of phospholamban, as shown previously with bovine calmodulin (Louis, C.F., and Jarvis, B. (1982) J. Biol. Chem. 257, 15187-15191). Thus, it appears that Ca2+-binding site I of calmodulin is at or near binding sites of calmodulin for TnI, TnT, and phospholamban. Analysis of the time-resolved fluorescence decay curves of I-EDANS-labeled calmodulin indicates a major component with a lifetime of 11.9 ns (+Ca2+), which accounts for 81% of the total fluorescence. The lifetime decreases slightly to 11.3 ns in the absence of Ca2+. Fluorescence anisotropy experiments indicate that I-EDANS-labeled CaM binds TnI with Kd = 6 x 10(-8) M in the presence of Ca2+. This study suggests that these single-site derivatives will be useful for characterizing a variety of calmodulin-receptor interactions because they lack ambiguities inherent in less specific labeling methods.     

Identification and characterization of calmodulin-binding proteins in islet secretion granules

The binding of 125I-calmodulin to intact secretion granules and protein gel blots of secretion granules from pancreatic islet tissue was examined. Binding of 125I-calmodulin to intact secretion granules was Ca2+-dependent and inhibited by the calmodulin inhibitors trifluoperazine and calmidazolium. Binding was inhibited by excess (200 nM) unlabeled calmodulin, but not by parvalbumin, a Ca2+-binding protein which has little sequence homology to calmodulin. In order to study the binding of calmodulin to specific secretion granule proteins, secretion granules were solubilized, and the solubilized proteins were resolved on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose, and incubated with 125I-calmodulin. Autoradiograms of the protein gel blots revealed the presence of three major calmodulin-binding proteins with approximate molecular weights of 73,000, 64,000, and 58,000. These proteins reversibly bound calmodulin in a calcium-dependent manner. Unlabeled calmodulin in the range of 0.1-1.0 nM competed with 125I-calmodulin for binding to these proteins, whereas troponin and parvalbumin were 100 and 1000-fold less effective, respectively. Trifluoperazine blocked binding to the granule proteins in a range of 10(-4) to 10(-5) M, and calmidazolium was effective between 10(-5) and 10(-6) M. Trypsin, at a concentration which did not lyse granules, markedly inhibited calmodulin binding to intact secretion granules. Protein blots from trypsin-treated granules showed that the three major calmodulin-binding proteins were absent. These results indicate that Ca2+-dependent calmodulin-binding proteins are present on the cytoplasmic surface of islet secretion granules and are consistent with the hypothesis that these proteins may play a role in secretion granule exocytosis.     

Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin

The Ca2+-binding component of troponin (TnC) and its proteolytic fragments containing Ca2+-binding sites I-III (TH1) or sites III and IV (TR2C) have been labeled with the fluorescent probes dansylaziridine (DANZ) at methionine 25 or 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid (AEDANS) at cysteine-98. These probes report binding of Ca2+ to the low and high affinity sites, respectively. Fluorescence changes as a function of [Ca2+] were measured for the free peptides, their complexes with troponin I + troponin T, and these complexes bound to actin-tropomyosin in the presence of Mg2+ and ATP with and without myosin. An apparent Hill coefficient of 1.0-1.1 has been obtained for the Ca2+-induced fluorescence changes in TnC, its fragments, and their ternary complexes regardless of the label used. When a ternary complex containing appropriately labeled TnC or its fragment is bound to the actin-tropomyosin complex, the Hill coefficient for the titration of the low affinity sites increases to 1.5-1.6 and further increases to greater than 2 in the presence of myosin. To interpret the apparent Hill coefficients, we used a model containing two binding sites and a single reporter of the conformational change. Hill coefficients between 1.0 and 1.2 can be obtained for the fluorescence change without true cooperativity in metal binding, depending on the mechanism of the fluorescence change; i.e. the contribution of the singly or doubly occupied species to the fluorescence change. A Hill coefficient between 1.2 and 2, however, always indicates cooperativity in binding independently of the mechanism. Thus, our finding that fluorescence titrations of Ca2+ binding to TnCDANZ bound to actin-tropomyosin exhibit a Hill coefficient of 1.5 in the absence of myosin and 2.4 in its presence indicates the existence of true positive cooperativity in metal binding to sites I and II. No cooperativity was observed for AEDANS-labeled complexes that reflect Ca2+-binding to the high affinity sites. Plots of the Ca2+ dependence of myosin ATPase activity activated by actin-tropomyosin in the presence of any of the troponin complexes used had apparent Hill coefficients of approximately 4. The higher value suggests cooperative interactions in the activation of ATPase beyond those involved in Ca2+-binding to the Ca2+-specific sites.     

Interaction between chicken gizzard caldesmon and tropomyosin

Chicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i.e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin- and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G.C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.     

Peptide binding by calmodulin and its proteolytic fragments and by troponin C

Calmodulin and troponin C exhibit calcium-dependent binding of 1 mol/mol of dynorphin. The dissociation constants of the complexes, determined in 0.20 N KC1-1.0 mM CaCI2, pH 7.3, are 0.6 microM for calmodulin, 2.4 microM for rabbit fast skeletal muscle troponin C, and 9 microM for bovine heart troponin C. Experiments with deletion peptides of dynorphin show that peptide chain length and especially charge affect the binding of the peptides by calmodulin. Dynorphin, but not mastoparan or melittin, inhibits adenosinetriphosphatase activity in a reconstituted rabbit skeletal muscle actomyosin assay. The inhibition is partially reversed by the addition of calmodulin or troponin C in the presence of calcium. Calmodulin also exhibits calcium-dependent binding of a synthetic peptide corresponding to positions 104-115 of rabbit fast skeletal muscle troponin I. Mastoparan is a tetradecapeptide from the vespid wasp having exceptional affinity for calmodulin, with Kd approximately 0.3 nM [Malencik, D.A., & Anderson, S.R. (1983) Biochem. Biophys. Res. Commun. 114, 50]. The addition of 1 mol/mol of mastoparan to the complex of calmodulin with dynorphin results in complete dissociation of dynorphin. Similar titrations of the skeletal muscle troponin C-dynorphin complex produce a gradual dissociation consistent with a dissociation constant of 0.2 microM for the troponin C-mastoparan complex. Fluorescence anisotropy measurements using the intrinsic tryptophan fluorescence of mastoparan X show strongly calcium-dependent binding by proteolytic fragments of calmodulin. binding by proteolytic fragments of calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium distribution of skeletal actin-tropomyosin-troponin states, determined by pyrene-tropomyosin fluorescence

Actin-tropomyosin-troponin has three structural states, but the functional properties of regulation can be explained with models having two functional states. As a step towards assigning functional properties to all the structural states, we examined fluorescent probes that monitor changes in troponin and tropomyosin. Tropomyosin labeled with pyrene-iodoacetamide is thought to reflect the transition to the most active state, whereas N-((2-iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I is thought to monitor the transition to any state other than the inactive state. The fraction of actin in an active state determined from pyrene excimer fluorescence agreed with that calculated from light-scattering measurements of myosin subfragment 1 (S1)-ADP to regulated actin in both the presence and absence of Ca2+ over a range of ionic strength conditions. The only exceptions were conditions where the binding of S1-ADP to actin was too strong to measure accurately. Pyrene-tropomyosin excimer fluorescence was Ca2+ dependent and so reflected the change in population caused by both Ca2+ binding and S1-ADP binding. Pyrene labeling of tropomyosin did not cause a large perturbation of the transition among states of regulated actin. Using pyrene-tropomyosin fluorescence we were able to extend the ionic strength dependence of the parameters describing the co-operativity of binding of S1-ADP to actin as low as 0.1 M. The probes on tropomyosin and troponin I had different responses to Ca2+ and S1-ADP binding. These different sensitivities can be explained by an intermediate between the inactive and active states of regulated actin.     

Effect of heparin on the expression of calmodulin-binding proteins in bull spermatozoa

A 125I-labelled calmodulin gel overlay procedure in the presence and the absence of Ca2+ was used to evaluate bull spermatozoa calmodulin-binding proteins. Frozen spermatozoa were thawed, washed and incubated for 6 h before being processed for SDS polyacrylamide gel electrophoresis and the 125I-labelled calmodulin gel overlay procedure. In non-incubated spermatozoa, up to 14 binding proteins were detected. Some exhibited greater calmodulin binding in the presence of Ca2+ while others exhibited greater binding when Ca2+ was absent. When heparin (2 micrograms/ml) was present in the incubation medium, a decrease in the calmodulin binding to the proteins of Mr 28,000 and 30,000 was detected in the presence of Ca2+ and EGTA. This effect of heparin was time- and dose-dependent and was increased by the presence of the acrosin inhibitor benzamidine. Sperm capacitation could thus be related to a decrease in the binding of calmodulin to these proteins.     

Mechanism of calmodulin recognition of the binding domain of isoform 1b of the plasma membrane Ca(2+)-ATPase: kinetic pathway and effects of methionine oxidation

Calmodulin (CaM) binds to a domain near the C-terminus of the plasma membrane Ca2+-ATPase (PMCA), causing the release of this domain and relief of its autoinhibitory function. We investigated the kinetics of dissociation and binding of Ca2+-CaM with a 28-residue peptide [C28W(1b)] corresponding to the CaM-binding domain of isoform 1b of PMCA. CaM was labeled with a fluorescent probe on either the N-terminal domain at residue 34 or the C-terminal domain at residue 110. Formation of complexes of CaM with C28W(1b) results in a decrease in the fluorescence yield of the fluorophore, allowing the kinetics of dissociation or binding to be detected. Using a maximum entropy method, we determined the minimum number and magnitudes of rate constants required to fit the data. Comparison of the fluorescence changes for CaM labeled on the C-terminal or N-terminal domain suggests sequential and ordered binding of the C-terminal and N-terminal domains of CaM with C28W(1b). For dissociation of C28W(1b) from CaM labeled on the N-terminal domain, we observed three time constants, indicating the presence of two intermediate states in the dissociation pathway. However, for CaM labeled on the C-terminal domain, we observed only two time constants, suggesting that the fluorescence label on the C-terminal domain was not sensitive to one of the kinetic steps. The results were modeled by a kinetic mechanism in which an initial complex forms upon binding of the C-terminal domain of CaM to C28W(1b), followed by binding of the N-terminal domain, and then formation of a tight binding complex. Oxidation of methionine residues in CaM resulted in significant perturbations to the binding kinetics. The rate of formation of a tight binding complex was reduced, consistent with the poorer effectiveness of oxidized CaM in activating the Ca2+ pump.     

Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence

Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

The effect of calmodulin inhibitors and the new cardiotonic drug DPI 201-106 on the formation of interprotein bonds in the cardiac troponin complex

Using the binding of labeled [125I]troponin C (TnC) to troponin I (TnI) and troponin (TnT) immobilized on a polyvinylchloride matrix, the Ca-dependent formation of interprotein bonds in the cardiac troponin complex and the effects of various drugs on the above reaction were studied. It has been found that in the absence of Ca2+ the dissociation constant, Kd, for the TnC-TnI complex in equal to (2.5 +/- 1.03).10(-7) M. In the presence of Ca2+ the number of binding sites increases twofold; the Kd value for the bonds formed thereby is (1.74 +/- 0.18).10(-7) M. The complex is stable to the effect of 5 M urea. TnC binding to immobilized TnT is nonspecific and is completely abolished by an addition of 5 M urea. DPI 201-106 used at concentrations up to 10(-3) M does not affect the Ca-dependent binding of TnC to TnI; trifluoperazine inhibits this interaction by 60%, whereas substance 48/80 inhibits the reaction by 50% when used at a concentration of 210 micrograms/ml. It is supposed that the compounds interacting with TnC affect, primarily, the cation-binding properties of troponin. These compounds can also inhibit the formation of interprotein bonds but only when used at much higher concentrations.     

Calcium binding to troponin C and troponin: effects of Mg2+, ionic strength and pH

Calcium binding to troponin C and troponin was examined by a metallochromic indicator method under various conditions to obtain a further understanding of the regulatory roles of these proteins in muscle contraction. Troponin C has four Ca binding sites, of which 2 sites have a high affinity of 4.5 X 10(6) M-1 for Ca2+ and the other 2 sites have a low affinity of 6.4 X 10(4) M-1 in a reaction medium consisting of 100 mM KCl, 20 mM MOPS-KOH pH 6.80 and 0.13 mM tetramethylmurexide at 20 degrees C. Magnesium also binds competitively to both the high and low affinity sites: the apparent binding constants are 1,000 M-1 and 520 M-1, respectively. Contrary to the claim by Potter and Gergely (J. Biol. Chem. 250, 4628-4633, 1975), the low affinity sites are not specific only for Ca2+. The high and low affinity sites of troponin C showed different dependence on the ionic strength: the high affinity sites were similar to GEDTA, while the low affinity sites were similar to calmodulin, which has a steeper ionic strength dependence than GEDTA. Ca binding to troponin C was not affected by change of pH between 6.5 and 7.2. Troponin I enhanced the apparent affinity of troponin C for Ca2+ to a value similar to that for troponin. Trifluoperazine also increased Ca binding to troponin C. Troponin has four Ca binding sites as does troponin C, but the affinities are so high that the precise analysis was difficult by this method. The apparent binding constants for Ca2+ and Mg2+ were determined to be 3.5 X 10(6) M-1 and 440 M-1, respectively, for low affinity sites under the same conditions as for troponin C, being independent of change in pH between 6.5 and 7.2. The competitive binding of Mg2+ to the low affinity sites of troponin is consistent with the results of Kohama (J. Biochem. 88, 591-599, 1980). The estimate for the high affinity sites is compatible with the reported results.     

Regulation of calmodulin binding to P-57. A neurospecific calmodulin binding protein

P-57 is a neural-specific calmodulin binding protein with novel calmodulin binding properties. P-57 exhibits higher affinity for calmodulin-Sepharose in the absence of free Ca2+ than in the presence of Ca2+ (Andreasen, T.J., Luetje, C.W., Heideman, W. & Storm, D.R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T.J., Andreasen, K.I. & Storm, D.R. (1985) J. Biol. Chem. 260, 10784-10788). In this study, the dissociation constants for P-57 and immunopurified 5-[[(iodoacetylamino)ethyl]-amino]-1-naphthalenesulfonic acid-labeled calmodulin (AEDANS-CaM) were determined under low and high ionic strength conditions. In the absence of added KCl, the dissociation constants for the P-57 X AEDANS-CaM complex were 2.3 X 10(-7) +/- 6 X 10(-8) M and 1.0 X 10(-6) +/- 3 X 10(-7) M in the presence and absence of excess Ca2+ chelator. The addition of KCl to 150 mM increased the Ca2+-independent and -dependent dissociation constants to 3.4 X 10(-6) +/- 9 X 10(-7) M and 3.0 X 10(-6) +/- 9 X 10(-7) M, respectively. The association of P-57 with AEDANS-CaM under low Ca2+ conditions was determined as a function of KCl concentrations. By taking into account the amount of P-57 found in brain and its affinity for calmodulin, it is concluded that most or all of the CaM would be complexed to P-57 in unstimulated cells. P-57 was phosphorylated by the Ca2+-phospholipid-dependent protein kinase (protein kinase C) with a phosphate:protein molar ratio of 1.3. Phosphoamino acid analysis demonstrated phosphorylation at a serine residue. CaM decreased the rate of phosphorylation of P-57 by protein kinase C, and phosphorylation prevented P-57 binding to calmodulin-Sepharose. P-57 was not phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase. It is proposed that P-57 binds and localizes calmodulin at specific sites within the cell and that free calmodulin is released locally in response to phosphorylation of P-57 by protein kinase C and/or to increases in intracellular free Ca2+. This regulatory mechanism, which appears to be specific to brain, would serve to decrease the response time for Ca2+-calmodulin-regulated processes.     

Biologically active fluorescent derivatives of spinach calmodulin that report calmodulin target protein binding

Spinach calmodulin (CaM) has been labeled at cysteine-26 with the sulfhydryl-selective probe 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) to produce MIANS-CaM. The interaction of MIANS-CaM with CaM binding proteins was studied by fluorescence enhancement accompanying the protein-protein interactions. MIANS-CaM bound to smooth muscle myosin light-chain kinase with a Kd of 9 nM, causing a 4.6-fold fluorescence enhancement. Caldesmon bound with a Kd of 250 nM, causing a 2-fold fluorescence enhancement. Calcineurin (CaN) bound to MIANS-CaM with a Kd less than 5 nM, causing an 80% increase in fluorescence. On the other hand, binding of the CaM antagonist drugs prenylamine and calmidazolium or the potent peptide antagonist melittin did not alter MIANS fluorescence. MIANS-CaM activated brain cGMP phosphodiesterase and CaN as effectively as unlabeled CaM. Spinach CaM was also labeled with three other sulfhydryl reagents, 6-acryloyl-2-(dimethylamino)naphthalene, (2,5-dimethoxy-4-stilbenyl)maleimide, and rhodamine X maleimide. CaN bound to the highly fluorescent rhodamine X maleimidyl-CaM with a Kd of 1.4 nM, causing a 25% increase in polarization. Both MIANS-CaM and rhodamine X-CaM were used to monitor the Ca2+ dependence of the interaction between CaM and CaN. Half-maximal binding occurred at pCa 6.7-6.8 in the absence of Mg2+, or at pCa 6.3 in the presence of 3 mM Mg2+. In both cases, the dependence of the interaction was cooperative with respect to Ca2+ (Hill coefficients of 1.7-2.0). Use of these fluorescent CaMs should allow accurate monitoring of CaM interactions with its target proteins and perhaps their localization within the cell.     

Photocrosslinking of calmodulin and/or actin to chicken gizzard caldesmon

The two sulfhydryl groups of chicken gizzard caldesmon were specifically labeled with a photoreactive crosslinker, benzophenone-maleimide, to study its interactions with calmodulin and/or actin. When incubated with F-actin caldesmon crosslinks to a single actin monomer; it can, however, crosslink to up to two calmodulin molecules in the presence, but not in the absence, of Ca2+. Thus caldesmon may have two calmodulin-binding sites, each containing, or being near, one of the two thiol residues. One of these two sites may also be adjacent to the actin-binding site. A calmodulin-binding fragment of caldesmon resulting from cyanogen bromide digestion crosslinks to a single calmodulin molecule, also in a Ca2+-dependent manner. Crosslinking of calmodulin to caldesmon does not prevent the latter from binding F-actin, suggesting that calmodulin and actin do not compete with each other for the same binding site(s) on the caldesmon molecule.     

Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.     

Static and kinetic studies of calmodulin and melittin complex.

Ca2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca(2+)-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca(2+)-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittin-calmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.     

Cardiac sarcoplasmic-reticulum calmodulin-binding proteins. Modulation of calmodulin binding to phospholamban by phosphorylation.

The gel-overlay technique with 125I-labelled calmodulin allowed the detection of several calmodulin-binding proteins of Mr 280 000, 150 000, 97 000, 56 000, 35 000 and 24 000 in canine cardiac sarcoplasmic reticulum. Only two calmodulin-binding proteins could be identified unambiguously. Among them, the 97 000-Mr protein that undergoes phosphorylation in the presence of Ca2+ and calmodulin, is likely to be glycogen phosphorylase. In contrast, the (Ca2+ + Mg2+)-activated ATPase did not appear to bind calmodulin under our experimental conditions. The second known calmodulin target is dephosphophospholamban, which migrates with an apparent Mr of 24 000. The dimeric as well as the monomeric form of phospholamban was found to bind calmodulin. Phospholamban shifts the apparent Kd of erythrocyte (Ca2+ + Mg2+)-activated ATPase for calmodulin, suggesting thus a tight binding of calmodulin to the proteolipid. Interestingly enough, phospholamban phosphorylation by either the catalytic subunit of cyclic AMP-dependent protein kinase or the Ca2+/calmodulin-dependent phospholamban kinase was found to inhibit calmodulin binding.     

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.