Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure

The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate processes, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength. The effect of 0.1 M KCl is to accelerate the slow process from 9.1 +/- 1.5 s-1 to 24 +/- 6.0 s-1 and to reduce the rate of the fast process from 650 s-1 to 240 +/- 50 s-1 at 25 degrees C. In the presence of 0.1 M KCl it was possible to determine activation parameters for the fast process: delta H# = 41 +/- 5 kJ mol-1 and delta S# = -63 +/- 17 J K-1 mol-1. These values are in good agreement with those obtained by 43Ca NMR. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the fast and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains. The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2+ dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the N-terminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.     

The interaction of melittin with calmodulin and its tryptic fragments

Melittin has been found to interact with both the N- and C-terminal half-molecules of calmodulin, as well as the intact molecule, in the presence of Ca2+. The interaction results in a major change in the microenvironment of Trp-19, which is in a more nonpolar, solvent-shielded, and immobilized microenvironment in the complex. The properties of Tyr-99 and Tyr-138 of calmodulin are altered by complex formation. From measurements of the efficiencies of radiationless energy transfer from Trp-19 to the nitro derivatives of Tyr-99 and/or Tyr-138, it is concluded that Trp-19 is located in proximity to the C-terminal lobe of calmodulin in the complex.     

Calcium binding to tryptic fragments of calmodulin

Fragments of scallop testis calmodulin were prepared by tryptic digestion. One peptide consisted of 75 amino acid residues from N-acetylalanine to lysine at position 75 (F12) and the other of 71 residues from aspartic acid at position 78 to C-terminal lysine (F34). Flow dialysis and equilibrium dialysis experiments revealed the existence of two Ca2+ binding sites in each fragment. Half-saturating concentrations of the Ca2+ titration curves were 11 microM for F12 and 3.2 microM for F34, and Hill coefficients were obtained as 1.14 and 1.84, respectively. The results indicate that the high-affinity sites for Ca2+ are located on the C-terminal region of the calmodulin. The sum of the two Ca2+ titration curves of F12 and F34 fits well to the curves of Ca2+ binding to intact calmodulin. This shows that the characteristic of Ca2+ bindings in intact calmodulin did not change after separation of the whole molecule into two domains, F12 and F34. The domains corresponding to F12 and F34 may exist independently from each other in the intact calmodulin molecule.     

Thermodynamics of Ca2+ binding to calmodulin and its tryptic fragments.

The binding of Ca2+ to calmodulin and its two tryptic fragments has been studied using microcalorimetry. The binding process is accompanied by the uptake or release of protons, depending on the ionic strength. With no added salt, the total enthalpy change for the binding of four calcium ions to calmodulin is -41 kJ mol-1 but in the presence of 0.15 mM KCl delta Htot is +17 kJ mol-1. The mode of binding of Ca2+ is also completely different with and without added salt. It is also shown that for the C-terminal fragment of calmodulin, TR2C, the drastic reduction in delta Gtot for the binding process on increasing the ionic strength is largely an enthalpic effect. Domain interactions in calmodulin are indicated by the fact that the sum of the enthalpies of calcium binding to the two tryptic fragments is not the same as the total binding enthalpy to calmodulin itself. The binding of Ca2+ to calmodulin has also been studied calorimetrically at different temperatures in the range 21-37 degrees C. delta Cp is large and negative in this interval.     

Effect of tryptic calmodulin fragments on guanylate cyclase activity from Paramecium tetraurelia

Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.     

Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase.     

Cyanogen bromide cleavage of bovine secretory component and its tryptic fragments

1. Five-domain bovine secretory component and its two-domain and three-domain tryptic fragments have been treated with cyanogen bromide. 2. N-terminal sequence analysis of the purified products showed that cleavage occurred within the disulphide bridged polypeptide loop of domain 2. The site lies within the region that binds IgM and IgA dimers. 3. The relative binding of the CNBr fragments to IgM has been measured and indicates that domains 1 and 3 are directly involved. 4. A possible role for domain 2 is less clear and domains 4 and 5 do not participate in binding.     

Proton nuclear magnetic resonance studies on the kinetics of tryptic fragments of calmodulin upon calcium binding

The kinetics of the Ca2+-dependent conformational change of the tryptic fragments F12 (residues 1-75) and F34 (residues 78-148) of calmodulin were studied by 1H-NMR. Resonances of two phenylalanines, 16 (or 19) and 65 (or 68), N epsilon, N epsilon, N epsilon-trimethyllysine-115 and tyrosine-138 were examined by the saturation-transfer technique or computer-aided line-shape simulation to obtain the rate of the conformational exchange between the Ca2+-free form and the Ca2+-bound form. The rates for F12 and F34 in the presence of 0.2 M KCl at 22 degrees C were 300-500 s-1 and 3-10 s-1, respectively. Activation parameters are as follows: Delta H not equal to = 11(+/- 2) kcal X M-1 and delta S not equal to = -9(+/- 5) cal X K-1 X M-1 for F12, and delta H not equal to = 16(+/- 2) kcal X M-1 and delta S not equal to = -2(+/- 5) cal X K-1 X M-1 for F34. These kinetic data for the conformational exchange are in agreement with those of Ca2+ dissociation from the binding sites obtained by 43Ca-NMR and stopped-flow fluorescence studies.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble 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)     

Agonist and antagonist properties of calmodulin fragments

Limited proteolysis of calmodulin with trypsin in the presence of ethylene glycol bis(beta-aminoethyl ether)-N, N,N',N'-tetracetic acid (EGTA) or Ca2+ was performed according to a modification of the method of Drabikowski et al. (Drabikowski, W., Kuznicki, J., and Grabarek, Z. (1977) Biochim. Biophys. Acta 485, 124-133). The resulting peptides were purified by reverse-phase high performance liquid chromatography. Tryptic digests in EGTA yielded peptides 1-106, 1-90, and 107-148 with yields of 9, 47, and 61%, respectively. The digests performed with Ca2+ yielded peptides 1-77 and 78-148 in 35 and 45% yield. Analysis by high performance liquid chromatography indicated that the purified fragments contained less than 0.1% contamination by calmodulin, thus allowing a definitive study of the ability of these fragments to activate, or interact with, calmodulin-regulated enzymes and anti-calmodulin drugs. Each of the fragments, except 107-148, bound to a phenothiazine affinity column in a Ca2+-dependent manner. Thus, calmodulin contains two interaction sites for phenothiazines: one on the NH2-terminal half (fragment 1-77) and one on the COOH-terminal half (fragment 78-148). None of the fragments activates the protein phosphatase, calcineurin, or prevents its stimulation by calmodulin, nor does any of the fragments stimulate Ca2+-dependent cAMP phosphodiesterase. A single cleavage in the middle of the calmodulin molecule results in the rapid dissociation of the two resultant fragments and a loss of ability to activate cAMP phosphodiesterase. One fragment, 78-148, interacts with phosphodiesterase and prevents its activation by calmodulin (Ki: 1.5 +/- 0.4 X 10(-6) M). The same fragment, 78-148, can fully activate phosphorylase kinase but with a lower affinity than calmodulin (Kuznicki, J., Grabarek, Z., Brzeska, H., Drabikowski, W., and Cohen, P. (1981) FEBS Lett. 130, 141-145). Thus, peptide 78-148 behaves as a calmodulin agonist or antagonist or as neither, depending on the enzyme under study.     

Structure of tryptic fragments of a neurotoxin from black widow spider venom

The N-terminal amino acid sequence of a neurotoxin from the venom of Latrodectus mactans tredecimguttatus (alpha-latrotoxin) was determined. Latrotoxin was subjected to the tryptic cleavage and total or partial amino acid sequences of 25 peptides were established. In total the tryptic fragments contained 252 amino acid residues. Essential structural information on cloning of the latrotoxin structural gene was obtained.     

Localization of hydrophobic sites in calmodulin and skeletal muscle troponin C studied using tryptic fragments: a simple method of their preparation

The exposure of hydrophobic sites on calmodulin, skeletal muscle troponin C and their tryptic fragments was investigated using Phenyl-Sepharose chromatography. A strong binding of both proteins and their fragments corresponding to the NH2-terminal halves of polypeptide chain of respective proteins in the presence of calcium ions was observed. Only a weak interaction with Phenyl-Sepharose or its lack was observed under these conditions for fragments corresponding to the COOH-terminal halves of calmodulin and troponin C, respectively. The elution of the samples from Phenyl-Sepharose column with ethylene glycol gradient allowed to compare relative hydrophobicity of both proteins and their fragments. The results show that hydrophobic properties of calmodulin and troponin C are virtually preserved in their fragments obtained as a result of their cleavage by trypsin in half. They also indicated that the exposure of hydrophobic residues caused by the binding of calcium ions takes place mainly in the NH2-terminal halves of polypeptide chains of both proteins. A simple method of purification of tryptic fragments of both proteins based on the difference in the strength of their interactions with Phenyl-Sepharose is described.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium measurements under the same conditions. These results are consistent with sequential dissociation of calcium from a narrow binding pocket inside which a single calcium ion can move fairly easily. Escape of calcium might be controlled by a structural compartment acting as a gate.     

Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.     

Carbon-13 NMR studies of the lysine side chains of calmodulin and its proteolytic fragments

ThepH-titration and dynamic behaviour of the seven lysine side chains in bovine calmodulin were studied by carbon-13 NMR. The amino groups of the calcium saturated protein and its proteolytic fragments TR₁C(1–75) and TR₂C (78–148) were dimethylated with carbon-13 labeled formaldehyde; this modification did not alter the protein's structure or its ability to activate the enzyme cyclic nucleotide phosphodiesterase. Tentative assignments for 5 out of the 7 dimethyl lysine resonances could be obtained by comparing spectra of the fully and partially modified protein, with those of the proteolytic fragments. ThepK_a values measured for calcium saturated calmodulin ranged between 9.5 (Lys 75) and 10.2 (Lys 13); two residues (Lys 94 and Lys 13) showed a biphasic titration curve suggesting their possible involvement in ion-pairs. The dynamic behavior of the lysine side chains was deduced from spin lattice relaxation measurements. All side chains were flexible and this was not influenced by the removal of calcium, or the addition of the calmodulin antagonist trifluoperazine. The latter data suggest that the lysine side chains are not directly involved in calmodulin's target binding sites.