Calcium-43 NMR studies of calcium-binding lysozymes and alpha-lactalbumins.

The calcium-binding properties of equine and pigeon lysozyme as well as those of bovine and human alpha-lactalbumin were investigated by 43Ca NMR spectroscopy. All proteins were found to contain one high-affinity calcium-binding site. The chemical shifts, line widths, relaxation times (T1 and T2), and quadrupole coupling constants for the respective 43Ca NMR signals were quite similar; this is indicative of a high degree of homology between the strong calcium-binding sites of these four proteins. The measured chemical shifts (delta approximately -3 to -7 ppm) and quadrupole coupling constants (chi approximately 0.7-0.8 MHz) are quite distinct from those observed for typical EF-hand calcium-binding proteins, suggesting a different geometry for the calcium-binding loops. The correlation times for bound calcium ions in these proteins were on the order of 4-8 ns, indicating that the flexibilities of these binding sites are limited. The apparent pKa values for the high-affinity sites ranged from 3.4 to 4.7, confirming the participation of carboxylate-containing residues in the coordination of the calcium ion. Competition experiments with EDTA showed that the affinities of these proteins for calcium follow the series bovine alpha-lactalbumin approximately human alpha-lactalbumin greater than pigeon lysozyme greater than equine lysozyme (KD approximately 5 x 10(-8) to 10(-6) M). Evidence for the existence of a second weak calcium-binding site (KD = 3 x 10(-3) M) was obtained for bovine alpha-lactalbumin, but not for the other proteins studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments

This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+X of the coordinating position of the calcium), Asp or Asn at position 3 (+Y), Gly at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix-loop-helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.     

Human alpha-fetoprotein and albumin: differences in zinc binding

The binding of zinc to both human alpha-fetoprotein (AFP) and albumin isolated from cord serum was studied by Sephadex G-50 gel-filtration chromatography. We found that the total number of binding sites for zinc on AFP and albumin were approximately 16 and 12, respectively. Both graphical analysis and the computer program 'LIGAND' indicate that there are at least two major classes of binding sites for both proteins. Both methods of analysis suggested that there are four to five high-affinity sites for zinc on AFP and only two to three similar sites on albumin. The affinity of zinc for AFP (dissociation constant, Kd, 6-8 X 10(-6) mol/l) was higher than for albumin (Kd, 1-3 X 10(-5) mol/l) for the high-affinity sites. The estimates for the zinc low-affinity binding sites were more uncertain, and several classes of low-affinity binding sites of different affinities might be present in both proteins. The results of our inhibition studies suggest that calcium, copper and lead might also bind with AFP at the zinc-binding sites.     

Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation

S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two alpha subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca(2+)-titration curves of Trp90 fluorescence for both the native protein and its Glu32 --> Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 --> Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K(1) =4 +/- 2 x 10(3) m(-1) (C-loops) and K(2) approximately 10(2) m(-1) (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid - a fluorescent probe of protein-surface hydrophobicity. S-Glutathionylation of the single cysteine residue (85) of the alpha subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous - four orders of magnitude - increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding to DeltaDeltaG = -6 +/- 1 kcal.mol(-1). A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.     

The calcium-binding site in the galactose chemoreceptor protein. Crystallographic and metal-binding studies

We have determined the relative affinities in solution for various metals which bind to the lone calcium-binding site of the D-galactose-binding protein which resembles the EF-hand loop. In order of affinity the metals are: Ca2+ approximately Tb3+ approximately Pb2+ greater than Cd2+ greater than Sr2+ greater than Mg2+ much greater than Ba2+. The binding affinity for calcium (Kd = 2 microM) and the slow off-rate determined for terbium (1 x 10(-3) s-1) and that the metal-binding site is unperturbed by sugar binding argue for a structural role. Furthermore, we have crystallographically refined the structure of the binding protein with the calcium substituted by cadmium, compared it with the calcium-bound structure, and found them to be identical. The results of these structural and solution studies support the hypothesis that for a given metal-binding loop, cation hydration energy, size, and charge are major factors contributing to binding affinity.     

Lead-binding properties of intestinal calcium-binding proteins

The bovine and chick vitamin D-induced intestinal calcium-binding proteins (CaBP) bind lead. Bovine CaBP binds 2 atoms of lead/molecule, and chick CaBP binds 4 atoms of lead per molecule and these values are identical to those for calcium binding. 45Calcium-displacement studies indicate significantly higher affinities for lead than for calcium for both proteins. All evidence indicates that lead is bound to the 4 high affinity calcium-binding sites on chick CaBP and to the corresponding 2 high affinity sites on bovine CaBP, and that binding of lead to sulfhydryl groups is, relatively, not significant. Calmodulin, troponin C, and oncomodulin also bind lead with high affinities and in preference to calcium, indicating that lead binding is a general property of proteins belonging to the troponin C superfamily of calcium-binding proteins.     

1H nuclear magnetic resonance study of the two calcium-binding sites of porcine intestinal calcium-binding protein

1H nuclear magnetic resonance has been employed to study the calcium-binding properties of the NH2- and COOH-terminal calcium-binding sites of the porcine intestinal calcium-binding protein. The protein was titrated with calcium in the presence of the chelator EDTA in order to determine the association constants of the protein for calcium relative to the known association constant of EDTA for calcium. The resulting data were compared with various models for the binding of calcium to two sites on the protein. Models were considered for which the two sites in the apoprotein have either intrinsically equal or unequal affinities for calcium. For each of these two cases, positive cooperativity, no cooperativity, and negative cooperativity were considered. The data fit best for the case of random binding to two independent sites with equivalent association constants of 1.0 +/- 0.1 X 10(7) M-1. The case of ordered binding to two sites with intrinsically different affinities, with concomitant positive affinity between the two sites so that the effective association constants were made equal, could not be mathematically excluded when only one protein NMR resonance is considered but can be shown to be implausible when the whole spectrum is considered.     

A flagellum-specific calcium sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.     

Spasmin and a Putative Spasmin Binding Protein(s) Isolated from Solubilized Spasmonemes1

ABSTRACT The amino acid composition and hydrophobicity scale (hydropathy) of calcium-binding proteins contained in the contractile spasmoneme of Carchesium polypinum was compared with other calcium-binding proteins from eukaryotes. Spasmins which may hind at most 4 calcium ions simultaneously and initiate spasmoneme contraction cooperatively belong to a super family of proteins including; centrin/caltractin and calmodulin. Based on chemical modification of tryptophan and methionine, these residues are involved in contraction but the spasmin proteins contain little or none of these amino acids. Based on this evidence, it is suggested that another, non-calcium binding protein(s) is involved in spasmoneme contraction.     

Calcium-binding properties of bovine factor X lacking the gamma-carboxyglutamic acid-containing region

In bovine protein C normal activation by the thrombin-thrombomodulin complex requires binding of calcium to one high affinity binding site, contained in a protein fragment that lacks the gamma-carboxyglutamic acid (Gla) region (Esmon, N. L., De Bault, L. E., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5548-5553). In this work, the calcium binding to and the conformational change induced by calcium in the corresponding Gla-domainless fragment of bovine factor X, prepared by limited proteolysis by chymotrypsin, were compared with the calcium-binding properties of Gla-domainless protein C. Equilibrium dialysis experiments demonstrated that the proteolytically modified factor X has one high affinity calcium ion-binding site with Kd = 180 microM, a value almost identical to the Kd for the binding of calcium to proteolytically modified protein C. Measurements of the rate of disulfide bond reduction by thioredoxin showed that the disulfide bonds of both factor X and protein C lacking the Gla domains were more rapidly reduced in the absence than in the presence of calcium. Thus, calcium binding induces a conformational change in both proteolytically modified proteins. Calcium binding to Gla-domainless protein C is accompanied by a quenching of the intrinsic tryptophan fluorescence and by changes in the CD spectrum, indicative of perturbation of the environment of aromatic amino acids by the metal ion. However, no such changes were observed with the proteolytically modified factor X. This difference may be due to the fact that one tryptophan residue (in position 84) is present in the light chain of the proteolytically modified protein C but none in the light chain of the modified factor X. The light chain of factor X has beta-hydroxyaspartic acid in position 64 which is homologous to the beta-hydroxyaspartic acid in position 71 in the light chain of protein C. Our results are compatible with the hypothesis that beta-hydroxyaspartic acid is involved in the Ca2+ ion binding.     

Search for fucose binding domains in recently sequenced hypothetical proteins using molecular modeling techniques and structural analysis

The crystal structure of a fucose-binding lectin from the bacteria Pseudomonas aeruginosa in complex with α-L-fucose has been recently determined. It is a tetramer; each monomer displays a nine-stranded, antiparallel, β-sandwiched arrangement and contains two calcium ions that mediate the binding of fucose in a recognition mode unique among protein-carbohydrate interactions. In search of this type of unique interactions in other newly discovered protein sequences, we have used molecular modeling techniques to predict and analyze the 3-D structures of some proteins, which exhibited reasonable degree of homology with the amino acid sequence of the bacterial protein. A BLAST search with the sequence of Pseudomonas aeruginosa as query in the non-redundant sequence database identified four proteins from different species, three organisms from bacteria and one from archaea. We have modeled the structures of these proteins as well as those of the complexes with carbohydrates and studied the nature of physicochemical forces involved in the complex formation both in presence and absence of calcium. The calcium-binding loops have been found to be highly conserved both in terms of primary and tertiary structures in these proteins, although a less acidic character is observed in Photorhabdus lectin due to the absence of two aspartic acid residues on the calcium-binding loop which also resulted in lower binding affinity. All these structures exhibited highly negative electrostatic environment in the vicinity of the calcium-binding loops which was essential for neutralizing the positive charges of two closely situated Ca⁺² ions. The comparison of the binding affinities of some monosaccharides other than fucose, e.g. mannose and fructose, showed higher binding energies confirming the fucose specificity of these proteins.     

Direct interaction of the calcium sensor protein synaptotagmin I with a cytoplasmic domain of the alpha1A subunit of the P/Q-type calcium channel.

Synaptotagmins are synaptic vesicle proteins containing two calcium-binding C2 domains which are involved in coupling calcium influx through voltage-gated channels to vesicle fusion and exocytosis of neurotransmitters. The interaction of synaptotagmins with native P/Q-type calcium channels was studied in solubilized synaptosomes from rat cerebellum. Antibodies against synaptotagmins I and II, but not IV co-immunoprecipitated [125I]omega-conotoxin MVIIC-labelled calcium channels. Direct interactions were studied between in vitro-translated [35S]synaptotagmin I and fusion proteins containing cytoplasmic loops of the alpha1A subunit (BI isoform). Gel overlay revealed the association of synaptotagmin I with a single region (residues 780-969) located in the intracellular loop connecting homologous domains II and III. Saturable calcium-independent binding occurred with equilibrium dissociation constants of 70 nM and 340 nM at 4 degrees C and pH 7.4, and association was blocked by addition of excess recombinant synaptotagmin I. Direct synaptotagmin binding to the pore-forming subunit of the P/Q-type channel may optimally locate the calcium-binding sites that initiate exocytosis within a zone of voltage-gated calcium entry.     

Calcium-binding proteins in the vorticellid spasmoneme

The proteins of the contractile spasmoneme from Vorticella convallaria, Carcheslium polypinum, and Zoothamnium geniculatum have been extracted in the detergent, sodium dodecyl sulfate (SDS), as well as urea and guanidine hydrochloride (GuCl). After SDS extraction, the molecular weight distribution of the proteins was examined by means of SDS-polyacrylamide gel electrophoresis. Significant amounts of material corresponding to the contractile proteins actin and tubulin are not present. The contractile organelles in the three species examined contain a group of closely related proteins of molecular weight near 20,000, which constitute a major part (40-60%) of the dry mass. The 20,000 mol wt proteins in Zoothamnium bind calcium with high affinity (pK congruent to 6) and are termed "spasmins." By means of urea polyacrylamide gel electrophorsis, it is demonstrated that in Carchesium and Zoothamnium certain spasmin components bind calcium even in the presence of 6 M urea. The binding of calcium in 6 M urea suggests a functional relationship between the spasmins and the calcium-binding proteins of striated muscle which behave similarly. The calcium binding in urea also indicates that the spasmins within a single spasmoneme have different calcium affinities, and this difference in calcium-binding properties may be an important factor in the physiological function of the organelle.     

Derivatives of blood coagulation factor IX contain a high affinity Ca2+-binding site that lacks gamma-carboxyglutamic acid

We have examined the calcium-binding properties and metal ion-dependent conformational changes of proteolytically modified derivatives of factor IX that lack gamma-carboxyglutamic acid (Gla) residues. Equilibrium dialysis experiments demonstrated that a Gla-domainless factor IX species retained a single high affinity calcium ion-binding site (Kd = 85 +/- 5 microM). Ca2+ binding to this site was accompanied by a decrease in intrinsic fluorescence emission intensity (Kd = 63 +/- 15 microM). These spectral changes were reversed upon the addition of EDTA. Titration with Sr2+ resulted in little change in fluorescence intensity below 1 mM, while titration with Tb3+ caused fluorescence changes similar to those observed with Ca2+. Tb3+ and Ca2+ appear to bind to the same site because tryptophan-dependent terbium emission was reduced by the addition of Ca2+. Similar results were obtained with a Gla-domainless factor IX species lacking the activation peptide. Gla domain-containing factor IX species exhibited fluorescence changes similar to those of the Gla-domainless proteins at low Ca2+, but an additional structural transition was found at higher Ca2+ concentrations (apparent Kd greater than 0.8 mM). Thus, the conformations of factor IX proteins are nucleated and/or stabilized by calcium binding to a high affinity site which does not contain Gla residues. The binding of Ca2+ to lower affinity Gla domain-dependent metal ion-binding sites elicits an additional conformational change. The strong similarities between these results and those obtained with protein C (Johnson, A. E., Esmon, N. L., Laue, T. M. & Esmon, C. T. (1983) J. Biol. Chem. 258, 5554-5560), coupled with the remarkable sequence homologies of the vitamin K-dependent proteins, suggest that the high affinity Gla-independent Ca2+-binding site may be a common feature of vitamin K-dependent proteins.     

Synthetic fragments of calmodulin calcium-binding site III. A test of the acid pair hypothesis

The acid pair hypothesis describing the interaction of calcium with the helix-loop-helix conformation of EF hands in calmodulin and related proteins predicts that these calcium-binding sites will have increased affinity for calcium if the anionic amino acid dentates in the loop region which interact directly with the cation are paired on the axial vertices of the resulting octahedral arrangement of chelating residues about the cation. As a test of this hypothesis, synthetic 33 residue analogs of bovine brain calmodulin calcium-binding site III have been prepared by the solid-phase method and analyzed for calcium affinity. The native sequence has a Kd of 735 microM for calcium and contains three anionic ligands which assume the +x, +y, and -z coordinates of the octahedral arrangement about the cation, thus precluding any pairing of the anionic ligands. This dissociation constant is 26 times weaker than that obtained from a synthetic analog of the sequentially homologous calcium-binding site III of rabbit skeletal TnC (Kd = 28 microM) which has four anionic ligands paired on the x and z axes. An analog of calmodulin site III with substitutions in the chelating residues at positions 1, 3, 5, 7, 9, and 12 of the 12-residue loop region to make these positions identical to those of rabbit skeletal troponin C site III decreased the calcium dissociation constant of the calmodulin peptide to 19 microM, similar to the troponin C peptide. Two synthetic analogs of calmodulin site III which contain three anionic ligands with two ligands paired on the x axis and two on the z axis have a Kd for calcium of 524 and 59 microM, respectively. This study provides strong support for and a better definition of the acid pair hypothesis and further demonstrates the usefulness of synthetic calcium-binding fragments in delineating the mechanism of calcium regulation of calmodulin and related proteins.     

Characterization of cardiac calsequestrin

Calsequestrin, a calcium-binding protein found in the sarcoplasmic reticulum of muscle cells, was purified from rabbit and canine cardiac and skeletal muscle tissue. The amino acid compositions and amino-terminal sequences of skeletal and cardiac calsequestrin from rabbit and dog were determined. The amino acid composition of the cardiac form was very similar to the skeletal form. The amino-terminal sequence of the cardiac form was homologous to, but not identical with, the amino-terminal sequence of the skeletal form of the protein. Few species differences in the amino-terminal sequences were observed. The calcium-binding capacity of the cardiac form was half the capacity of the skeletal form although the affinities of the two forms of calsequestrin for Ca2+ were similar (Kd = 1 mM). Calcium binding to the cardiac form induced structural changes in the protein as determined by circular dichroism and intrinsic fluorescence spectroscopy. The alpha-helical content of cardiac calsequestrin increased from 3.5% to 10.9% upon binding calcium, while the intrinsic fluorescence of the protein increased 14%. Potassium ions also affected the conformation of cardiac calsequestrin.     

Stoichiometric and electrostatic characterization of calcium binding to native and lipid-substituted adenosinetriphosphatase of sarcoplasmic reticulum

The stoichiometry of calcium binding to specific sites (i.e., those producing enzyme activation) was found to be 8-10 nmol/mg protein in native sarcoplasmic reticulum vesicles, and 13.9-15.4 nmol/mg of ATPase purified by non-ionic detergent solubilization and anion exchange chromatography. Parallel measurements of phosphoenzyme yielded levels of 4.0-4.9 and 6.0-7.7 nmol/mg of protein in the two preparations, respectively, demonstrating that each 115 kDa ATPase chain includes one catalytic site and two calcium binding sites. The apparent association constant, K = (6 +/- 2) X 10(5) M-1, and the binding cooperativity, nH = 1.9, were unchanged when measurements were carried out with native sarcoplasmic reticulum vesicles and when the membrane surface charge was altered by lipid substitution with phosphatidylcholine or phosphatidylserine, at neutral pH in the presence of 10 mM MgCl2 and 80 mM KCl. On the other hand, the apparent association constant was increased in the absence of Mg2+ or, to a lesser extent, in the absence of monovalent cations. It was also observed that the cooperative character of the calcium binding isotherms was reduced in low ionic-strength media. Analysis of the electrostatic effects indicates that the calcium-binding domain is shielded from the membrane phospholipid surface charge by virtue of its location within the ATPase protein. The effects of various electrolytes are attributed to monovalent-cation binding in the calcium-binding domain. The apparent loss of cooperativity of the calcium binding isotherms at low ionic strength is attributed to a progressive displacement of the titration curve which is minimal at low degrees of saturation and becomes larger at higher degrees of saturation. This behavior is described quantitatively by the progressive effect of calcium binding on an electrostatic potential generated by localized protein charge densities within, or near, the calcium-binding domain.     

Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis

Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.     

A grafting approach to obtain site-specific metal-binding properties of EF-hand proteins

The EF-hand calcium-binding loop III from calmodulin was inserted with glycine linkers into the scaffold protein CD2.D1 at three locations to study site-specific calcium binding properties of EF-hand motifs. After insertion, the host protein retains its native structure and forms a 1:1 metal-protein complex for calcium and its analog, lanthanum. Tyrosine-sensitized Tb3+ energy transfer exhibits metal binding and La3+ and Ca2+ compete for the metal binding site. The grafted EF-loop III in different environments has similar La3+ binding affinities, suggesting that it is largely solvated and functions independently from the host protein.     

Interaction of copper nd zinc cations with calcium-binding proteins

Interactions of the calcium binding proteins, parvalbumin from cod muscles, alpha-lactalbumin from cow milk and calmodulin from bovine brain, with Cu2+ and Zn2+ ions have been studied by intrinsic fluorescence and microcalorimetry methods. It was revealed that parvalbumin binds one Cu2+ ion per molecule with association constant from 10(5) to 10(6) M-1. Zn2+ ions seem to compete for the same site which does not coincide with the two Ca2+ and Mg2+ binding sites. alpha-Lactalbumin contains from 2 to 4 Cu2+ and Zn2+ binding sites, the number and affinities of which depend on Ca2+ concentration. Calmodulin has similar Cu2+ and Zn2+ binding sites. The binding of Cu2+ and Zn2+ ions to parvalbumin and alpha-lactalbumin changes the shape and position of their thermal denaturation transitions. The results obtained together with the literature data show that the ability to interact with Cu2+ and Zn2+ ions is a property inherent to many calcium-binding proteins, which may play a physiological role for some of them.