Thermodynamic analysis of heparin binding to human antithrombin.

The binding of heparin to human antithrombin III (ATIII) was investigated by titration calorimetry (TC) and differential scanning calorimetry (DSC). TC measurements of homogeneous high-affinity pentasaccharide and octasaccharide fragments of heparin in 0.02 M phosphate buffer and 0.15 M sodium chloride (pH 7.3) yielded binding constants of (7.1 +/- 1.3) x 10(5) M-1 and (6.7 +/- 1.2) x 10(6) M-1, respectively, and corresponding binding enthalpies of -48.3 +/- 0.7 and -54.4 +/- 5.4 kJ mol-1. The binding enthalpy of heparin in phosphate buffer (0.02 M, 0.15 M NaCl, pH 7.3) was estimated from TC measurements to be -55 +/- 10 kJ mol-1, while the enthalpy in Tris buffer (0.02 M, 0.15 M NaCl, pH 7.3) was -18 +/- 2 kJ mol-1. The heparin-binding affinity was shown by fluorescence measurements not to change under these conditions. The 3-fold lower binding enthalpy in Tris can be attributed to the transfer of a proton from the buffer to the heparin-ATIII complex. DSC measurements of the ATIII unfolding transition exhibited a sharp denaturation peak at 329 +/- 1 K with a van 't Hoff enthalpy of 951 +/- 89 kJ mol-1, based on a two-state transition model and a much broader transition from 333 to 366 K. The transition peak at 329 K accounted for 9-18% of the total ATIII. At sub-saturate heparin concentrations, the lower temperature peak became bimodal with the appearance of a second transition peak at 336 K. At saturate heparin concentration only the 336 K peak was observed. This supports a two domain model of ATIII folding in which the lower stability domain (329 K) binds and is stabilized by heparin.     

Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.     

Thermodynamic comparison of PNA/DNA and DNA/DNA hybridization reactions at ambient temperature

The thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T and their complementary PNA and DNA sequences were determined from isothermal titration calorimetry (ITC) measurements at ambient temperature. For the PNA/DNA hybridization reactions, the binding constants range from 1.8 x 10(6)M(-1)for PNA(TT)/DNA to 4.15 x 10(7)M(-1)for PNA(GA)/DNA and the binding enthalpies range from -194 kJ mol(-1)for PNA(CG)/DNA to -77 kJ mol(-1)for PNA(GT)/DNA. For the corresponding DNA/DNA binding reactions, the binding constants range from 2.9 x 10(5)M(-1)for DNA(GT)/DNA to 1.9 x 10(7)M(-1)for DNA(CC)/DNA and the binding enthalpies range from -223 kJ mol(-1)for DNA(CG)/DNA to -124 kJ mol(-1)for DNA(TT)/DNA. Most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences resulting from smaller entropy changes in the PNA/DNA hybridization reactions. van't Hoff enthalpies and extrapolated Delta G values determined from UV melting studies on the duplexes exhibited closer agreement with the ITC binding enthalpies and Delta G values for the DNA/DNA duplexes than for the PNA/DNA duplexes.     

Interactions of cytochrome c with DNA at glassy carbon surface

Cyclic voltammetry (CV) was used to investigate the interactions of Cytochrome c (Cyt c) with deoxyribonucleic acid (DNA) at glassy carbon (GC) electrodes. The results indicate that there are strong interactions between Cyt c and DNA. The binding constant (k(A)) and binding free energy (Delta(r)G) of Cyt c with dsDNA are (1.69+/-0.38) x 10(5) L.mol(-1) and -(29.76+/-0.56) kJ.mol(-1), respectively; and those of Cyt c with ssDNA are (3.35+/-0.50) x 10(5) L.mol(-1) and -(31.49+/-0.37) kJ.mol(-1), respectively. The binding sites are achieved to be 3.3 bp per Cyt c molecule with dsDNA and 4.0 nucleotides (ssDNA) binding one Cyt c molecule. This experiment affords a valid method for investigating the interactions between DNA and proteins by electrochemical techniques.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3

The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.     

Spectroscopic studies of interaction of chlorobenzylidine with DNA

Electronic absorbance and fluorescence titrations are used to probe the interaction of chlorobenzylidine with DNA. The binding of chlorobenzylidine to DNA results in hypochromism, a small shift to a longer wavelength in the absorption spectra, and emission quenching in the fluorescence spectra. These spectral characteristics suggest that chlorobenzylidine binds to DNA by an intercalative mode. This conclusion is reinforced by fluorescence polarization measurements. Scatchard plots constructed from fluorescence titration data give a binding constant of 1.3 x 10(5) M(-1) and a binding site size of 10 base pairs. This indicates that chlorobenzylidine has a high affinity with DNA. The intercalative interaction is exothermic with a Van't Hoff enthalpy of -143 kJ/mol. This result is obtained from the temperature dependence of the binding constant. The interaction of chlorobenzylidine with DNA is affected by the pH value of the solution. The binding constant has its maximum at pH 3.0. Upon binding to DNA, the fluorescence from chlorobenzylidine is quenched efficiently by the DNA bases and the fluorescence intensity tends to be constant at high concentrations of DNA when the binding is saturated. The Stern-Volmer quenching constant obtained from the linear quenching plot is 1.6 x 10(4) M(-1) at 25 degrees C. The measurements of the fluorescence lifetime and the dependence of the quenching constant on the temperature indicate that the fluorescence quenching process is static. The fluorescence lifetime of chlorobenzylidine is 1.9 +/- 0.4 ns.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 1.85 x 10(-5) M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (K(D) = 1.71 x 10(-7) M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K(-1) mol(-1). There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.     

DNA-binding cytotoxic alkaloids: comparative study of the energetics of binding of berberine, palmatine, and coralyne

Deoxyribonucleic acid is the site of storage and retrieval of genetic information through interaction with proteins and other small molecules. In the present study, the interaction of two natural cytotoxic protoberberine plant alkaloids, berberine and palmatine, and a synthetic derivative, coralyne, with mammalian herring testis DNA was investigated using a combination of isothermal titration calorimetry, differential scanning calorimetry, and optical melting experiments to characterize the energetics of their binding. The binding constants of these alkaloids to DNA under identical conditions were evaluated from the UV melting data, and the enthalpy of binding was elucidated from isothermal titration studies. The binding constants of berberine, palmatine, and coralyne to DNA were found to be 1.15 x 10(4), 2.84 x 10(4), and 3.5 x 10(6) M(-1) at 20 degrees C in buffer of 20 mM [Na+]. Parsing of the free energy change of the interaction observed into polyelectrolytic and nonpolyelectrolytic components suggested that although these alkaloids are charged, the major contributor of about 75% of the binding free energy arises from the nonpolyelectrolytic forces. The binding in case of palmatine and coralyne was predominantly enthalpy driven with favoring smaller entropy terms, while that of berberine was favored by both negative enthalpy and positive entropy changes. Temperature dependence of the binding enthalpies determined from ITC studies in the range 20-40 degrees C was used to calculate the binding-induced change in heat capacity (DeltaC(o)(p)) values as -117, -135, and -157 cal/mol K, respectively, for berberine, palmatine, and coralyne. Taken together, the results suggest that the DNA binding of the planar synthetic coralyne is stronger and thermodynamically more favored compared to the buckled natural berberine and palmatine.     

Entropic drive in the sarcoplasmic reticulum ATPase interaction with Mg2+ and Pi.

Thermodynamic quantities for the binding of Mg2+ (in the presence of Ca2+) and Pi (in the presence of Mg2+ and absence of Ca2+) to sarcoplasmic reticulum ATPase were determined from isothermal titration calorimetry measurements at 25 degrees C. Mg2+ and Pi are involved in reversal of the ATPase hydrolytic reaction, and their interactions with the ATPase are conveniently studied under equilibrium conditions. We found that the Mg2+ binding reaction is endothermic with a binding constant (Kb) = 142 +/- 4 M(-1), a binding enthalpy of 180 +/- 3 kJ mol(-1), and an entropy contribution (TdeltaSb) = 192 +/- 3 kJ mol(-1). Similarly, Pi binding is also an endothermic reaction with Kb = 167 +/- 17 M(-1), deltaHb = 65.3 +/- 5.4 kJ mol(-1), and TdeltaSb = 77.9 +/- 5.4 kJ mol(-1). Our measurements demonstrate that the ATPase can absorb heat from the environment upon ligand binding, and emphasize the important role of entropic mechanisms in energy transduction by this enzyme.     

Thermodynamic analysis of binding and protonation in DOTAP/DOPE (1:1): DNA complexes using isothermal titration calorimetry

A better understanding of the nature of the interaction between various cationic lipids used for gene delivery and DNA would lend insight into their structural and physical properties that may modulate their efficacy. We therefore separated the protonation and binding events which occur upon complexation of 1:1 DOTAP (1,2-dioleoyl-3-trimethylammonium propane):DOPE (1,2-dioleoylphosphatidylethanolamine) liposomes to DNA using proton linkage theory and isothermal titration calorimetry (ITC). The enthalpy of DOPE protonation was estimated as -45.0+/-0.7 kJ/mol and the intrinsic binding enthalpy of lipid to DNA as +2.8+/-0.3 kJ/mol. The pK(a) of DOPE was calculated to shift from 7.7+/-0.1 in the free state to 8.8+/-0.1 in the complex. At physiological ionic strength, proton linkage was not observed upon complex formation and the buffer-independent binding enthalpy was +1.0+/-0.4 kJ/mol. These studies indicate that the intrinsic interaction between 1:1 DOTAP/DOPE and DNA is an entropy-driven process and that the affinities of cationic lipids that are formulated with and without DOPE for DNA are controlled by the positive entropic changes that occur upon complex formation.     

The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 degrees C to -10 degrees C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mol bp)(-1). Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4) J K(-1) (mol bp)(-1) and -5.0(+/-0.5) kJ (mol bp)(-1), respectively.     

Divalent ion-binding properties of the two avian beta-parvalbumins

Birds express three parvalbumins, one alpha isoform and two beta isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T-cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca2+- and Mg2+-binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepes-buffered saline, ATH binds Ca2+ with apparent microscopic binding constants of 2.4 +/- 0.2 x 10(8) and 1.0 +/- 0.1 x 10(8) M(-1). The corresponding values for CPV3-C72S are substantially lower, 4.5 +/- 0.5 x 10(7) and 2.4 +/- 0.2 x 10(7) M(-1), a 1.9-kcal/mol difference in binding free energy. Thus, the beta-parvalbumin lineage displays a spectrum of Ca2+-binding affinity, with ATH and the mammalian beta isoform at the high- and low-affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca2+ affinity, CPV3-C72S exhibits increased affinity for Mg2+, relative to ATH. Whereas the latter displays Mg2+-binding constants of 2.2 +/- 0.2 x 10(4) and 1.2 +/- 0.1 x 10(4) M(-1), CPV3-C72S yields values of 5.0 +/- 0.8 x 10(4) and 2.1 +/- 0.3 x 10(4) M(-1).     

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.     

The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid

The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.     

Association of the AB and CD-EF domains from rat alpha- and beta-parvalbumin

Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.