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.     

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.     

The interaction between calmodulin and microtubule proteins. IV. Quantitative analysis of the binding between calmodulin and tubulin dimer

We reported previously that calmodulin binds to tubulin in a Ca2+-dependent manner, thereby inhibiting microtubule assembly. In this work, we quantitatively investigated the binding between calmodulin and tubulin by applying two analytical methods. One was the frontal analysis using affinity chromatography originally developed by Kasai and Ishii (J. Biochem. 84, 1061-1069, 1978). The use of tubulin-Sepharose columns gave a dissociation constant of 4.0 microM. The other was the equilibrium gel filtration developed by Hummel and Dreyer (Biochim. Biophys. Acta 63, 532-534, 1962). This method using a Sephadex G-100 column provided a dissociation constant of 3.5 microM under the same medium conditions as in the frontal analysis, and it was found that 2 mol calmodulin could bind to 1 mol tubulin. Furthermore, the frontal analysis method was convenient for studies on the effect of temperature and ionic strength on the binding. Upon elevating the temperature, the dissociation constant increased. Increase in the ionic strength also increased the dissociation constant.     

Calcium binding to tubulin

Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites (1.56 +/- 0.38 per tubulin dimer) with a dissociation constant of (4.86 +/- 0.12).10(-6) M and low-affinity binding sites (5.82 +/- 0.50 per tubulin dimer) with a dissociation constant of (6.4 +/- 0.4).10(-5) M. In the presence of 6.10(-5) M MgSO4, we found 0.64 +/- 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of (4.7 +/- 0.5).10(-6) M and 1.2 +/- 0.2 sites per dimer with a dissociation constant of (3.5 +/- 0.4).10(-5) M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium-binding sites.     

Ion binding to calmodulin

Summary Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca²⁺ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca²⁺ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca^2– binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.Abbreviations CaBP calcium-binding protein - ICaBP the vitamin D-dependent intestinal Cat+binding protein - S-100 the glial S-100 protein - RLC the phosphorylatable myosin regulatory light chain - CaM calmodulin - Pa parvalbumin - TnC troponin C - TnI troponin I - Hepes N-2-hydroxyethylpipezarine, N-2-ethane-sulfonic acid - W7 N-(6-Aminohexyl)-5-chloro-l-Naphtalene sulfonamide - SDS sodium dodecyl sulfate - NMR nuclear magnetic resonance     

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.     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Calcium-dependent and -independent binding of soybean calmodulin isoforms to the calmodulin binding domain of tobacco MAPK phosphatase-1

The recent finding of an interaction between calmodulin (CaM) and the tobacco mitogen-activated protein kinase phosphatase-1 (NtMKP1) establishes an important connection between Ca(2+) signaling and the MAPK cascade, two of the most important signaling pathways in plant cells. Here we have used different biophysical techniques, including fluorescence and NMR spectroscopy as well as microcalorimetry, to characterize the binding of soybean CaM isoforms, SCaM-1 and -4, to synthetic peptides derived from the CaM binding domain of NtMKP1. We find that the actual CaM binding region is shorter than what had previously been suggested. Moreover, the peptide binds to the SCaM C-terminal domain even in the absence of free Ca(2+) with the single Trp residue of the NtMKP1 peptides buried in a solvent-inaccessible hydrophobic region. In the presence of Ca(2+), the peptides bind first to the C-terminal lobe of the SCaMs with a nanomolar affinity, and at higher peptide concentrations, a second peptide binds to the N-terminal domain with lower affinity. Thermodynamic analysis demonstrates that the formation of the peptide-bound complex with the Ca(2+)-loaded SCaMs is driven by favorable binding enthalpy due to a combination of hydrophobic and electrostatic interactions. Experiments with CaM proteolytic fragments showed that the two domains bind the peptide in an independent manner. To our knowledge, this is the first report providing direct evidence for sequential binding of two identical peptides of a target protein to CaM. Discussion of the potential biological role of this interaction motif is also provided.     

Kinetics of calmodulin binding to calcineurin

Calcineurin (CaN) binds Ca(2+)-saturated calmodulin (CaM) with relatively high affinity; however, an accurate steady-state K(d) value has not been determined. In this report, we describe, using steady-state and stopped-flow fluorescence techniques, the rates of association and dissociation of Ca(2+)-saturated CaM from CaN heterodimer (CaNA/CaNB) and CaNA only. The rate of Ca(2+)/CaM association was determined to be 4.6 x 10(7) M(-1)s(-1). The rate of Ca(2+)/CaM dissociation from CaN was slower than previously reported and was approximately 0.0012 s(-1). In preparations of CaNA alone (no regulatory CaNB subunit), the dissociation rate was slowed further to 0.00026 s(-1). From these data we calculate a K(d) for binding of Ca(2+)-saturated CaM to CaN of 28 pM. This K(d) is significantly lower than previously reported estimates of approximately 1 nM and indicates that CaN is one of the highest affinity CaM-binding proteins identified to date.     

Nucleotide-dependent bisANS binding to tubulin.

Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.     

Rhizoxin binding to tubulin at the maytansine-binding site

The binding of rhizoxin, a potent inhibitor of mitosis and in vitro microtubule assembly, to porcine brain tubulin was studied. Tubulin possesses one binding site for rhizoxin per molecule with a dissociation constant (Kd) of 1.7.10(-7) M. Ansamitocin P-3, a homologue of maytansine, was a competitive inhibitor of rhizoxin binding, with an inhibition constant of 1.3.10(-7) M. Vinblastine also inhibited rhizoxin binding, but was not fully competitive, and the inhibition constant was 2.9.10(-6) M. In contrast, both rhizoxin and ansamitocin P-3 were potent inhibitors of vinblastine binding. Rhizoxin inhibited tau-promoted tubulin assembly, but it, differing from vinblastine, did not induce tubulin aggregation into spirals, even at a concentration as high as 2.10(-5) M. In addition, rhizoxin strongly inhibited vinblastine-induced tau-dependent tubulin aggregation. Rhizoxin binding to tubulin was completely independent from colchicine binding. These effects resemble those of maytansine. The results suggested that rhizoxin binds to the maytansine-binding site and that the binding sites of rhizoxin and vinblastine are not the same.     

Colchicine binding to an oligomer of tubulin

An oligomeric form of tubulin present in microtubule protein prepared from mammalian brain, the 36S double ring containing tau protein, is reported to bind colchicine. Colchicine binds to each individual 6S tubulin subunit in the 36S ring without apparent effect on quarternary structure. The colchicine-oligomer complex forms by colchicine binding directly to the tubulin ring; alternatively, complexes formed by colchicine with 6S tubulin subunits associate in the presence of tau protein to form the colchicine-oligomer complex.     

Thermodynamics of zinc binding to rhodanese

The binding of zinc ion (Zn²⁺) to rhodanese at two pH values was studied by microcalorimetry and the free energy, enthalpy, and entropy changes determined. Binding exhibited rather large endothermic enthalpy changes quite similar to those observed for zinc-model compound interactions. The large positive entropy changes which accompany binding appear to be a feature common to Zn²⁺-apocarbonic anhydrase systems as well. The correlations between Zn²⁺ interaction with model compounds resembling protein side chains and the thermodynamic values obtained for Zn²⁺-protein interactions suggest that endothermic enthalpies of binding should commonly be observed under slightly acidic to basic conditions. It is found that commercial rhodanese binds Zn²⁺ with moderate to weak affinity by a process that is entropy driven much like that of other Zn²⁺-protein interactions.     

Drug-protein interactions: binding of chlorpromazine to calmodulin, calmodulin fragments, and related calcium binding proteins

The quantitative binding of a phenothiazine drug to calmodulin, calmodulin fragments, and structurally related calcium binding proteins was measured under conditions of thermodynamic equilibrium by using a gel filtration method. Plant and animal calmodulins, troponin C, S100 alpha, and S100 beta bind chlorpromazine in a calcium-dependent manner with different stoichiometries and affinities for the drug. The interaction between calmodulin and chlorpromazine appears to be a complex, calcium-dependent phenomenon. Bovine brain calmodulin bound approximately 5 mol of drug per mol of protein with apparent half-maximal binding at 17 microM drug. Large fragments of calmodulin had limited ability to bind chlorpromazine. The largest fragment, containing residues 1-90, retained only 5% of the drug binding activity of the intact protein. A reinvestigation of the chlorpromazine inhibition of calmodulin stimulation of cyclic nucleotide phosphodiesterase further indicated a complex, multiple equilibrium among the reaction components and demonstrated that the order of addition of components to the reaction altered the drug concentration required for half-maximal inhibition of the activity over a 10-fold range. These results confirm previous observations using immobilized phenothiazines [Marshak, D.R., Watterson, D.M., & Van Eldik, L.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6793-6797] that indicated a subclass of calcium-modulated proteins bound phenothiazines in a calcium-dependent manner, demonstrate that the interaction between phenothiazines and calmodulin is more complex than previously assumed, and suggest that extended regions of the calmodulin molecule capable of forming the appropriate conformation are required for specific, high-affinity, calcium-dependent drug binding activity.     

Spectroscopic and kinetic features of allocolchicine binding to tubulin

Allocolchicine is a structural isomer of colchicine in which colchicine's tropone C ring is replaced with an aromatic ester. In spite of the structural differences between the two ligands, the association parameters for both molecules binding to tubulin are quite similar. The association constant for allocolchicine binding to tubulin was determined by fluorescence titration to be 6.1 x 10(5) M-1 at 37 degrees C, which is about a factor of 5 less than that of the colchicine-tubulin association. In particular, analysis of the kinetics of the association of allocolchicine with tubulin yielded nearly equivalent activation parameters for the two ligands. The activation energy of the allocolchicine binding reaction was found to be 18.4 +/- 1.5 kcal/mol, which is only slightly less than the activation energy for colchicine binding to tubulin. This finding argues against conformational flexibility of the C ring as the structural feature of colchicine responsible for the slow kinetics of colchicinoid-tubulin binding reactions. Tubulin binding promote a dramatic enhancement of allocolchicine fluorescence. Unlike colchicine, the emission energy and intensity of the tubulin-bound allocolchicine fluorescence can be mimicked by solvent, and a general hydrophobic environment for the ligand binding site is indicated. The excitation spectrum of the protein-bound species, however, is shown to possess two bands which center at higher and lower energy than the energy maximum of the spectrum of the ligand in apolar solvents, indicating that properties of the colchicine binding site in addition to a low dielectric constant contribute to the fluorescence of the bound species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of substrate binding to the chaperone SecB

The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of alpha-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal mol(-1) K(-1), respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

Covalent binding of acetaldehyde to tubulin: evidence for preferential binding to the alpha-chain

The covalent binding of [14C]acetaldehyde to purified beef brain tubulin was characterized. As we have found for several other proteins, tubulin bound acetaldehyde to form both stable and unstable adducts. Unstable adducts (Schiff bases) were stabilized, and rendered detectable, by treating incubated reaction mixtures with the reducing agent sodium borohydride. In short-term incubations, the majority of the adducts formed were unstable, but the percentage of total adducts that were stable gradually increased with time. Stable adduct formation was greatly increased by the inclusion of sodium cyanoborohydride in reaction mixtures (reductive ethylation). When reaction mixtures were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the alpha- and beta-chains of the heterodimeric tubulin molecule, the alpha-chain of free tubulin, but not intact microtubules, was the preferential site of stable adduct formation under both reductive and nonreductive conditions. Denaturation studies showed that the native tubulin conformation was necessary for the alpha-chain to show enhanced reactivity toward acetaldehyde. Competition binding studies showed that alpha-tubulin could effectively compete with beta-tubulin and bovine serum albumin for a limited amount of acetaldehyde. Unstable acetaldehyde adducts with free tubulin or microtubules did not exhibit alpha-chain selectivity. Analysis of reaction mixtures indicates that lysine residues are the major group of the protein participating in adduct formation. These data indicate that the alpha-chain of free tubulin is the preferential site of stable acetaldehyde-tubulin adduct formation. Further, these data raise the possibility that alpha-tubulin may be a selective target for acetaldehyde adduct formation in cellular systems.     

The growth cone cytoskeleton. Glycoprotein association, calmodulin binding, and tyrosine/serine phosphorylation of tubulin

Cytoskeletons were prepared from the growth cones of neonatal rat forebrains and were utilized to explore several aspects of growth cone function. The cytoskeletal fraction retained about 50% of total growth cone protein, was highly enriched in tubulin, and constituted an interconnected lattice of 10-25 nm homogeneous particles. The cytoskeleton appeared to be a target for Ca2+ signaling since it contained the majority of growth cone calmodulin-binding polypeptides which featured prominently an Mr 135,000 component. Most of the growth cone glycoproteins were at least partially associated with the cytoskeleton, thus suggesting the possibility of a transmembrane coupling mechanism which allows for communication between the cytoskeleton and the external surface of the growth cone. The cytoskeleton was also endowed with one or more protein kinases which phosphorylated endogenous and exogenous tubulin achieving a stoichiometry of 9-13 mol of phosphate/mol of substrate dimer. Interestingly, the site of tubulin phosphorylation included tyrosine as well as serine residues providing a possible target for the action of neuronal tyrosine kinases such as growth factor receptors or oncogene products. Finally, a comparison between cytoskeletal preparations from growth cones and from mature synaptosomes revealed several differences in glycoprotein association, calmodulin binding, and protein phosphorylation, evidently reflecting maturational events which might underlie relevant aspects of synaptogenesis.