High Affinity Calcium Binding Sites Research Articles

Characterization of factor VII association with tissue factor in solution. High and low affinity calcium binding sites in factor VII contribute to functionally distinct interactions

Protein-phospholipid as well as protein-protein interactions may be critical for tight binding of the serine protease factor VIIa (VIIa) to its receptor cofactor tissue factor (TF). To elucidate the role of protein-protein interactions, we analyzed the interaction of VII/VIIa with TF in the absence of phospholipid. Binding of VII occurred with similar affinity to solubilized and phospholipid-reconstituted TF. Lack of the gamma-carboxyglutamic acid (Gla)-domain (des-(1-38)-VIIa) resulted in a 10- to 30-fold increase of the Kd for the interaction, as did blocking the Gla-domain by Fab fragments of a specific monoclonal antibody. These results suggest that the VII Gla-domain can participate in protein-protein interaction with the TF molecule per se rather than only in interactions with the charged phospholipid surface. Gla-domain-independent, low affinity binding of VII to TF required micromolar Ca2+, indicating involvement of high affinity calcium ion binding sites suggested to be localized in VII rather than TF. Interference with Gla-domain-dependent interactions with TF did not alter the TF. VIIa-dependent cleavage of a small peptidyl substrate, whereas the proteolytic activation of the protein substrate factor X was markedly decreased, suggesting that the VIIa Gla-domain not only participates in the formation of a more stable TF. VIIa complex but contributes to extended substrate recognition.

Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX

Factor IX is a blood clotting protein that contains three regions, including a gamma-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF-like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel beta-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp28, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the beta-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.

A circular dichroic study of Cu(II) binding to bovine α-lactalbumin

The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine α-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed charge-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.

Calcium binding by monosulfate esters of taurochenodeoxycholate

The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.

Interaction of potassium and magnesium with the high affinity calcium-binding sites of the sarcoplasmic reticulum calcium-ATPase.

The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane.

Protein S and C4b-Binding Protein: Components Involved in the Regulation of the Protein C Anticoagulant System

The protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF) epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 gamma-carboxy glutamic acid residues in the vitamin K-dependent domain, a beta-hydroxylated aspartic acid in the first EGF-like domain and a beta-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical alpha-chains and one beta-chain. The alpha- and beta-chains are linked by disulphide bridges. The cDNA cloning of the beta-chain showed the alpha- and beta-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the beta-chain to contain the single protein S binding site on C4BP, whereas each of the alpha-chains contains a binding site for the complement protein, C4b. As C4BP lacking the beta-chain is unable to bind protein S, the beta-chain is required for protein S binding, but not for the assembly of the alpha-chains during biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of haemophilia B patients with mutations in the two calcium binding domains of factor IX: importance of a β‐OH Asp 64→Asn change

The polymerase chain reaction procedure (PCR) coupled with direct sequencing has been used to screen a panel of haemophilia B patients. This analysis has identified, amongst others, several mutations in the functionally important gla and type B EGF domains of factor IX, both of which are known to bind calcium. Type B EGF domains are widely distributed in proteins; located within these domains are highly conserved amino acid residues important for the formation of a high-affinity calcium binding site. One prominent feature of these domains is a highly conserved beta-hydroxylated Asp or Asn residue. Of particular interest is the identification of one patient, with a substitution of the beta-hydroxy Asp-64 residue normally present in factor IX for Asn. This change results in a functionally defective factor IX molecule with altered calcium binding properties. To explain the functional abnormality caused by this substitution of one amino acid residue for another which is commonly found at the equivalent position in other proteins with type B EGF domains, we propose the existence of additional conserved residues within this domain, which are important for calcium binding, and which correlate with whether the beta-hydroxylated residue is Asp or Asn.

Tertiary structure and energy coupling in Ca2+-pump system

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8-9 A and the average distance from the Ca2+ sites to CrATP is about 18 A. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2(+)-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.

Novel type of very high affinity calcium-binding sites in beta-hydroxyasparagine-containing epidermal growth factor-like domains in vitamin K-dependent protein S.

Vitamin K-dependent protein S is shown to contain four very high affinity Ca2(+)-binding sites. The number of sites and their affinities were determined from Ca2+ titration in the presence of the chromophoric chelator Quin 2. In 0.15 M NaCl, pH 7.5, the four macroscopic binding constants are K1 greater than or equal to 1 x 10(8) M-1, K2 = 3 +/- 2 x 10(7) M-1, K3 = 4 +/- 2 x 10(6) M-1, and K4 = 9 +/- 4 x 10(5) M-1. At low ionic strength, the corresponding values are K1 greater than or equal to 2 x 10(9) M-1, K2 = 9 +/- 4 x 10(8) M-1, K3 = 2 +/- 1 x 10(8) M-1, and K4 = 9 +/- 4 x 10(7) M-1. To localize the Ca2(+)-binding sites, protein S was subjected to proteolysis using lysyl endopeptidase. This yielded a 20-21-kDa fragment which comprised the third and fourth epidermal growth factor (EGF)-like domains and remained high affinity Ca2(+)-binding site(s). The susceptibility of the EGF-like domains to proteolysis increased when Ca2+ was removed from protein S indicating that the Ca2+ binding is important for the stability and/or conformation of the EGF domains. Three of the four EGF-like domains in protein S contain beta-hydroxyasparagine. In each of these domains there is a cluster of three or four negatively charged amino acid residues which are likely to contribute to the extraordinary high Ca2+ affinity. From sequence homology it is suggested that this novel type of high affinity Ca2(+)-binding site is present in several other proteins, e.g. in the EGF-like domains in the low sensity lipoproteins receptor, thrombomodulin, the Notch protein of Drosophila melanogaster, and transforming growth factor beta 1-binding protein.

The human alpha 2-macroglobulin receptor contains high affinity calcium binding sites important for receptor conformation and ligand recognition.

The receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) was purified recently, and its binding of ligand was shown to depend on calcium ions (Moestrup, S. K., and Gliemann, J. (1989) J. Biol. Chem. 264, 15574-15577). This paper shows that the 440-kDa human placental alpha 2MR is a cysteine-rich glycoprotein with high affinity calcium binding sites important for receptor conformation; and the relationship between Ca2+ concentration and receptor function is presented. Autoradiography showed 45Ca2+ binding to the 440-kDa alpha 2MR blotted onto nitrocellulose from a sodium dodecyl sulfate-polyacrylamide gel. alpha 2MR immobilized on nitrocellulose in the absence of sodium dodecyl sulfate bound 45Ca2+ in the presence of 5 mM Mg2+, and 2-3 microM unlabeled Ca2+ was required to displace half of the bound 45Ca2+. The calcium concentration dependence showed upward concave Scatchard plots, and the number of binding sites was estimated to be approximately eight/alpha 2MR molecule. Binding of calcium did not change in the pH range 6.5-8.0 but decreased at lower pH values. Addition of Ca2+ to the medium was necessary for receptor binding of the alpha 2-macroglobulin-trypsin complex, and half of the maximal binding capacity was obtained with about 16 micrograms Ca2+ at pH 7.8. The requirement for calcium was increased at lower pH values, and half of the maximal 125I-alpha 2M-trypsin binding was obtained with about 30-40 microM Ca2+ at pH 7.0. Monoclonal antibodies were produced against alpha 2MR, and one of them distinguished between the Ca2(+)-occupied and nonoccupied forms. Like Ca2+, Sr2+ and Ba2+ elicited ligand binding affinity and competed for binding with 45Ca2+ in the order Ca2+ greater than Sr2+ greater than Ba2+. In conclusion, calcium ions bind specifically to alpha 2MR with high affinity, and it is likely that several sites on the alpha 2MR molecule have to be occupied to elicit the conformation recognizing the ligand.

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

It has been suggested that epidermal growth factor-like (EGF-like) domains, containing conserved carboxylate residues, are responsible for the high-affinity calcium binding exhibited by a number of vitamin K-dependent plasma proteins involved in the control of the blood coagulation cascade. These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S. To test this hypothesis we have expressed the first EGF-like domain from human factor IX (residues 46-84) using a yeast secretion system, and examined calcium binding to the domain. Using 1H-NMR to measure a calcium-dependent shift assigned to Tyr69 we have detected a high-affinity calcium binding site (Kd = 200-300 microM). We suggest that other EGF-like domains of this type may have similar calcium binding properties. In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites.

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.

Characterization of the gene for mp20: a Drosophila muscle protein that is not found in asynchronous oscillatory flight muscle.

A Drosophila melanogaster gene encoding a muscle specific protein was isolated by differential screening with RNA from primary cultures of myotubes. The gene encodes a 20-kD protein, muscle protein 20 (mp20), that is not detected in the asynchronous oscillatory flight muscles, but is found in most, if not all, other muscles (the synchronous muscles). The sequence of the protein, deduced from the DNA, contains two regions of 12 amino acids with significant similarity to high-affinity calcium-binding sites of other proteins. This protein is easily extracted from the contractile apparatus and thus does not seem to be a tightly bound structural component. The gene (located in polytene region 49F 9-13) is unique in the D. melanogaster genome and yields two transcripts, 1.0 and 0.9 kb long. The levels of the two transcripts are regulated differently during development, yet the coding regions of the two transcripts are identical.

Calcium binding to the epidermal growth factor homology region of bovine protein C.

A high affinity calcium binding site that is independent of the gamma-carboxyglutamic acid-rich amino-terminal region, has been demonstrated in bovine protein C, as well as in the other vitamin K-dependent proteins (except prothrombin) involved in blood coagulation. gamma-Carboxyglutamic acid-independent calcium binding in protein C is required for its rapid activation by the thrombin-thrombomodulin complex. We have now isolated a Ca2+-binding fragment from a tryptic digest of bovine protein C. The isolated fragment contains the two domains that are homologous to the epidermal growth factor precursor from the light chain of protein C, and a small disulfide bound peptide derived from the heavy chain. The isolated fragment bound 1 mol of Ca2+/mol of protein with a dissociation constant (Kd) of approximately 1 x 10(-4) M. This is similar to the Kd previously determined for binding of a single Ca2+ ion to protein C lacking the gamma-carboxyglutamic acid region. Immunochemical evidence indicated that Ca2+ binding induced a conformational change both in protein C lacking the gamma-carboxyglutamic acid region and in the isolated fragment.

Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid).

Osteonectin is a prominent noncollagenous protein of developing bone. A 2150-base-pair cDNA coding for osteonectin, isolated from a bovine bone cell lambda gt11 expression library, was sequenced and identified by comparison with protein sequence data. The nucleotide sequence predicts that osteonectin contains 304 amino acids, including a 17-residue signal peptide. Analysis of the deduced protein sequence suggests that the secreted protein contains at least four distinct structural domains. An acidic region at the amino terminus of the protein appears to be a potential hydroxyapatite-binding site. This is followed by a second domain, rich in cysteine, that shows sequence homology with cysteine-rich domains in turkey ovomucoid and other serine proteinase inhibitors. Two sequences homologous with central calcium-binding loops of "EF hands" and thus having potential to be high-affinity calcium-binding sites are located in two other domains within the carboxyl-terminal half of the protein. Finally, the osteonectin sequence shows near identity (greater than 90%) with another protein, SPARC (secreted protein, acidic and rich in cysteine), secreted by mouse parietal endoderm. These data suggest that osteonectin, a protein present in bone and other selected tissues, is a multifunctional protein.

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The two high affinity calcium binding sites of the cardiac (Ca2+ + Mg2+)-ATPase have been identified with the use of Eu3+. Eu3+ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H2O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca2+ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6-8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.

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 MgSO 4, 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 α- and β-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the α-subunit, and major fragments of 31 kDa and 22 kDa from the β-subunit. The high-affinity calcium-binding sites were detecte 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.

Calcium-dependent interaction between the epidermal growth factor precursor-like region of human protein C and a monoclonal antibody.

Protein C, like the other vitamin K-dependent plasma proteins that participate in blood coagulation, except prothrombin, has at least one high affinity calcium-binding site that is independent of gamma-carboxyglutamic acid. Calcium binding to this site is required for activation of protein C by the thrombin-thrombomodulin complex. In an attempt to localize this calcium-binding site, we subjected protein C to limited tryptic digestion. A monoclonal antibody that recognizes a calcium-dependent epitope both in intact protein C, in gamma-carboxyglutamic acid-domainless protein C, and in activated protein C, was used to isolate a fragment from the tryptic digest. The fragment was derived from the light chain of protein C and consisted of the two domains that are homologous to the epidermal growth factor precursor. Half-maximal binding of the intact protein and of the isolated fragment by the antibody occurred at 100-200 microM Ca2+. The results suggest the presence of a Ca2+-binding site in the epidermal growth factor homology region of protein C.

Calcium binding to untreated and dephosphorylated porcine neurofilaments

The calcium-binding properties of untreated and in vitro dephosphorylated neurofilaments were determined by partition centrifugation. Scatchard plot analysis of the binding data indicated that each type of neurofilament contained both high and low affinity calcium-binding sites. The number of calcium-binding sites per unit consisting of three 140,000, three 107,000 and eight 62,000 molecular weight subunits was 4 sites with Kd=4.1 μM and 126 sites with Kd=293 μM for untreated neurofilaments. The in vitro dephosphorylated neurofilaments contained 8 sites with Kd=15 μM and 54 sites with Kd-332 μM, per unit.

Localization of site-specific probes on the Ca-ATPase of sarcoplasmic reticulum using fluorescence energy transfer.

Highly reactive sulfhydryls, previously labeled with an iodoacetamide spin label on the Ca-ATPase of sarcoplasmic reticulum, were labeled with the fluorescent probe, 5-(2-[iodoacetyl)amino)ethyl)aminonaphthalene-1-sulfonic acid (IAEDANS), without loss of enzymatic activity. We have selectively measured the apparent distance of the more reactive site, relative to other site-specific probes at both the nucleotide and the high affinity calcium binding sites. Fluorescence energy transfer efficiencies from the donor IAEDANS to two acceptors: fluorescein 5'-isothiocyanate or 2',3'-O-(2,4,3-trinitrophenyl)adenosine monophosphate, situated at or near the nucleotide site, were measured using fluorescence lifetimes and yields. Fluorescence on polyacrylamide gels shows that the IAEDANS and fluorescein 5'-isothiocyanate labels are both associated with the B tryptic fragment. The energy transfer measurements are consistent with distances of 56 and 68 A between IAEDANS and these respective binding sites. On the other hand, energy transfer measurements using the lanthanide, praseodymium (Pr3+), as an acceptor indicate that IAEDANS is located 16-18 A from the binding site(s) of this calcium analog. Pr3+ is shown to be a good analog for calcium binding to the high affinity sites on the enzyme since it competitively displaces calcium, as evidenced by the reversal of the specific calcium-dependent intrinsic fluorescent signal and inactivation of ATPase activity, over the same narrow range in Pr3+ concentration where energy transfer is observed. Our observations suggest that the portion of the B fragment spanning the cytoplasmic portion of the ATPase is folded onto the A fragment, bringing the IAEDANS label in close proximity to the high affinity calcium binding domain.