High-resolution proton and laser photochemically induced dynamic nuclear polarization NMR studies of cation binding to bovine alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) is a calcium binding protein that also binds Mn(II), lanthanide ions, A1(III), Zn(II), Co(II). The structural implications of cation binding were studied by high-resolution proton (200 MHz) NMR and photochemically induced dynamic nuclear polarization (CIDNP) spectroscopy. Marked changes were observed in the NMR spectra of the apoprotein upon addition of a stoichiometric amount of calcium to yield Ca(II)-alpha-LA, manifested particularly in ring current shifted aliphatic peaks and in several shifts in the aromatic region, all of which were under slow exchange conditions. The CIDNP results showed that two surface-accessible tyrosine residues, assigned as Tyr-18 and -36, became inaccessible to the solvent upon addition of 1:1 Ca(II) to apo-alpha-lactalbumin, while Tyr-103 and Trp-104 remained completely accessible in both conformers. The proton NMR spectra of apo-alpha-LA and A1(III)-alpha-LA were extremely similar, which was also consistent with intrinsic fluorescence results [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. The paramagnetic cation Mn(II) bound to the strong calcium binding site on apo-alpha-LA but also to the weak secondary Ca(II) binding site(s) on Ca(II)-alpha-LA. It was also found that Co(II) bound to some secondary sites on Ca(II)-alpha-LA that overlapped the weak calcium site. All of the lanthanide shift reagents [Pr(III), Eu(III), Tb(III), Dy(III), Tm(III), Yb(III)] bound under slow exchange conditions; their relative affinities for apo-alpha-lactalbumin from competitive binding experiments were Dy(III), Tb(III), and Pr(III) greater than Ca(II) greater than Yb(III).     

Binding of ions and hydrophobic probes to alpha-lactalbumin and kappa-casein as determined by analytical affinity chromatography

The technique of analytical affinity chromatography was extended to characterize binding of ions and hydrophobic probes to proteins. Using the immobilized protein mode of chromatography, alpha-lactalbumin and kappa-casein were covalently attached to 200-nm-pore-diameter controlled-pore glass beads and accommodated for high-performance liquid chromatography. The existence of a high affinity binding site (Kdiss = 0.16 microM) (site I) for calcium ion in alpha-lactalbumin was confirmed by chromatography of [45Ca2+]. In addition, chromatography of the hydrophobic probes, 1-(phenylamino)-8-naphthalene-sulfonate (ANS)2 and 4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate (bis-ANS) indicated that Ca2+ bound to a second site (presumably the zinc site or site II) with weaker affinity. Dissociation constants obtained for apo-alpha-lactalbumin were about 80 microM for ANS and 4.7 microM for bis-ANS in the absence of sodium ion. Addition of Ca2+ initially caused a reduction in surface hydrophobicity (lowered affinity for the probe dyes) followed by an increase at higher Ca2+ concentrations (greater than 0.5 mM), suggesting that occupancy of site II restores an apo-like conformation to the protein. Moreover, the effect of Zn2+ was similar to that observed in the higher Ca2+ concentration range, whereas Na+ apparently bound to site I. A calcium binding site of moderate affinity also exists in kappa-casein (Kdiss = 15.6 microM). A cluster of negative charges, probably including the orthophosphate group, most likely comprise this binding site. By preventing self-association, analytical affinity chromatography permits microscale characterization of ligand equilibria in proteins that are unaffected by protein-protein interactions.     

Alpha-lactalbumin binding to membranes: evidence for a partially buried protein

The self-incorporation of apo-alpha-lactalbumin (alpha-LA) into single unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine was demonstrated by column chromatographic analyses on Sephadex G-200 (10 mM Tris-HCl, pH 7.4, 26-28 degrees C) and by intrinsic fluorescence emission of SUV-bound alpha-LA. It was shown that apo-alpha-LA slowly incorporated into the DMPC vesicle bilayer after equilibrating different mixtures of protein and SUV for several hours. The intrinsic fluorescence properties of the bound apo-alpha-LA were altered only slightly (lambda maxem = 333 nm vs. 337 nm in aqueous solution). The large blue shift in apo-alpha-LA fluorescence in solution induced by monovalent cations, such as Na(I), was almost completely prevented when apo-alpha-LA was membrane bound. Furthermore, the addition of calcium caused a slow conversion from apo-alpha-LA to Ca(II)-alpha-LA by a mechanism consistent with passive diffusion of Ca(II) into the bilayer interior to the (buried) calcium binding site. The release of Ca(II)-alpha-LA from the membrane is discussed with reference to alpha-LA release from the smooth endoplasmic reticulum in vivo.     

Physiological roles of zinc and calcium binding to alpha-lactalbumin in lactose biosynthesis

Bovine apo-alpha-lactalbumin was shown to be severalfold more efficient than its calcium conformer as a cofactor in lactose biosynthesis. This rate enhancement was manifested in a 3.5-fold increase in Vmax, with no differences in Km(app) between the two alpha-lactalbumin forms. In the presence of zinc, which shifts Ca(II)-alpha-lactalbumin toward the "apo-like" conformation [Musci, G., & Berliner, L.J. (1985) Biochemistry 24, 3852-3856], the catalytic rate constant for lactose synthesis was identical for both the Ca(II) and apo conformers. Activity measurements at different temperatures, on the other hand, confirmed that calcium is important in stabilizing the protein (alpha-lactalbumin) against thermal denaturation. The stabilizing effect of calcium was independent of the presence of Zn(II), i.e., of the protein conformation. The physiological implications of these results are discussed.     

Intramolecular distance measurements in alpha-lactalbumin

The distance between the calcium site (site I) and the zinc site (site II) in alpha-lactalbumin was estimated from Forster energy-transfer measurements between donor Eu(III) [or Tb(III)] at site I and acceptor Co(II) at site II to be 11.5 +/- 1.5 A. Intersite distances were also measured between the bis-ANS [4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate]] binding locus and cobalt at site II (13.6 +/- 1.0 A), between bis-ANS and a fluorescein moiety covalently bound to Met-90 (33.5 +/- 3.0 A), and between Met-90 (fluorescein) and cobalt at site II (16.7 +/- 1.0 A). The apparent Kd for cobalt binding to site II agreed well with the value measured previously by intrinsic fluorescence [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. A Zn(II) titration of Eu(III)-alpha-lactalbumin reconfirmed that both sites I and II can be occupied simultaneously [Musci, G., & Berliner, L. J. (1985) Biochemistry 24, 3852-3856], since the lanthanide fluorescence was unaffected.     

Manganese (II) electron spin resonance and cadmium-113 nuclear magnetic resonance evidence for the nature of the calcium binding site in alpha-lactalbumins

Bovine and goat alpha-lactalbumins were substituted with 113Cd(II) or Mn(II) at the strong calcium site [Murakami, K., Andree, P.J., & Berliner, L.J. (1982) Biochemistry 21, 5488-5494] and studied by 113 Cd NMR and electron spin resonance. The 113Cd chemical shifts were in the -80 to -85 ppm range vs. Cd(ClO4)2, which was almost identical with that found for several nearly octahedral (oxygen-coordinated) calcium binding proteins such as calmodulin, parvalbumin, and troponin C. The electron spin resonance spectra of bound Mn(II)-alpha-lactalbumin complexes at 9 or 35 GHz were also confirmatory of a highly symmetric (cubic) environment around the Mn(II) with only slight distortions. The near identity of this site in alpha-lactalbumin to those of calcium binding proteins containing an "EF hand domain" was remarkable despite the absence of such a domain sequence in the alpha-lactalbumin structure.     

4,4'-Bis[8-(phenylamino)naphthalene-1-sulfonate] binding to human thrombins: a sensitive exo site fluorescent affinity probe

The binding of the fluorescent probe 4,4'-bis[8-(phenylamino)naphthalene-1-sulfonate] (bis-ANS) to human alpha- and gamma-thrombins was investigated. Bis-ANS binds in a 1:1 complex to both forms of the enzyme, with Kd = 14.8 +/- 2.2 microM and 5.8 +/- 1.0 microM for alpha- and gamma-thrombin, respectively, at pH 7.0 [25 mM tris(hydroxymethyl)aminomethane, 0.15 M NaC1]. Fluorescence changes upon complexation included a considerable (approximately 30-nm) blue shift in the fluorescence emission maximum as well as a dramatic increase in the fluorescence emission intensity: a 70-fold enhancement was observed with alpha-thrombin vs. a approximately 220-fold enhancement with gamma-thrombin. Proflavin was not displaced upon bis-ANS binding. The unknown thrombin effectors ATP, Ca(II)ATP, Co(III)ATP, phosphate, and pyrophosphate bound with enhancement of the fluorescence of the bis-ANS-alpha-thrombin complex. The two inhibitors benzamidine and p-chlorobenzylamine as well as heparin caused decreases in bis-ANS-thrombin fluorescence: valerylamidine had no effect on the fluorescence of the bis-ANS-thrombin complex. Kinetic measurements with two chromogenic substrates, S-2238 and S-2160, indicated that bis-ANS acts as a partial noncompetitive inhibitor of thrombin amidase activity. The kinetic evidence combined with the ligand binding results suggests that bis-ANS does not overlap the catalytic site. The fluorophore ANS complexed with equal affinity to both alpha- and gamma-thrombins (Kd = 24 +/- 4 microM); however, the gamma-thrombin-ANS complex emission at 470 nm was enhanced 26% more than that for the alpha form.     

Metal ion binding to alpha-lactalbumin species

A strong cation (calcium) binding site has been demonstrated to exist in several alpha-lactalbumin species; bovine, goat, human, and guinea pig. A metal ion induced conformational change occurs, resulting in a unique (10-14-nm) blue shift and relative quenching of Trp fluorescence for all species. Calcium ion binding to the alpha-lactalbumins yielded dissociation constants (Kdiss consistently in the 10(-10)--10(-12) M range, while Mn(II) binding was in the 20-30 microM range. Independent determinations of these cation binding equilibria were made by ESR measurements of free unliganded Mn(II) in titrations with the bovine species. One strong site (Kdiss = 30.5 microM) was found, which correlated directly with the fluorescence-associated cation binding, plus three weaker sites (Kdiss = 1.1, 5.0, and 5.0 mM, respectively). Several lanthanides as well as Mg(II) were found to displace Mn(II) from the strong site on bovine alpha-lactalbumin (as monitored by ESR) and to cause the identical fluorescence changes as found for Ca(II) and Mn(II) above. The importance of measuring these equilibria by both fluorescence and ESR was borne out by demonstrating the potential errors in estimating dissociation equilibria by the fluorescence method alone. Also, the errors in estimating Kdiss for samples containing partially metal bound apo-alpha-lactalbumin are described as well as rapid, sensitive methods for estimating the extent of metal-free protein and correctly accounting for residual bound metal in equilibrium calculations.     

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,1'-bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate acts as an inhibitor of lipoprotein lipase and competes for binding with apolipoprotein CII

Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1'-bis(anilino)-4,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant Kd of 0.10-0.26 microm and by a relatively high on rate constant kass = 2.0 x 10(4) m(-1) s(-1) and a slow off-rate with a dissociation rate constant kdiss = 1.2 x 10(-4) s(-1). The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII.     

The binding of Mn2+ to bovine plasma protein C, des(1-41)-light chain protein C, and activated des(1-41)-light chain activated protein C

The binding isotherms of Mn2+ to bovine plasma protein C (PC), des(1-41)-light chain protein C (GDPC), and activated GDPC (GDAPC) have been measured. PC contains 14-16 total Mn2+ binding sites, a value that is reduced to approximately 7-8 in the presence of NaCl. The average Kd of the latter sites is 230 +/- 30 microM. Upon removal of a 41-residue peptide from the amino terminus of the light chain of PC, and, concomitantly, all of the gamma-carboxyglutamic acid residues, the resulting protein, GDPC, possesses a single Mn2+ site of Kd = 120 +/- 20 microM. Activation of GDPC to GDAPC results in a slight lowering of the Kd for the single Mn2+ binding site to 53 +/- 8 microM, a value that is essentially unchanged in the presence of monovalent cations, a competitive inhibitor of the enzyme, or an active site directed affinity label. The Mn2+ on GDAPC is displaced by Ca2+, suggesting that the protein binding site for these two divalent cations is the same. These studies establish that Mn2+ is a suitable spectroscopic probe for the Ca2+ binding site of GDAPC, and that the divalent cation site is separate from the monovalent cation site(s) and the active site of the enzyme.     

Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis.

Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.     

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

The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine alpha-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed change-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.     

An electron paramagnetic resonance study of bovine alpha-lactalbumin-metal ion complexes

alpha-lactalbumin has at least three distinct cation binding regions: a Ca(II)-Gd(III) site, a Cu(II)-Zn(II) site and a VO2+ site as observed from electron paramagnetic resonance (EPR) studies of complexes with the bovine protein. Gadolinium, which bound to the calcium site of the protein with a subnanomolar dissociation constant, yielded EPR spectra at 9.5 GHz (X-band) that exhibited features from g = 8 to g = 2. At 35 GHz (Q-band) the central fine structure transition (Ms = 1/2----Ms = -1/2) gave a well-defined powder pattern. The zero-field splitting was large, as reflected in the second-order splitting of the central fine structure transition of about 1 kG. There was also evidence for additional, low affinity binding site(s) for Gd(III). Addition of either Zn(II) or Al(III) did not affect the amplitudes or positions of the bound Gd(III) EPR spectrum. The Cu(II)-alpha-lactalbumin complex gave a typical axially symmetric spectrum (g parallel = 2.260, g perpendicular = 2.056, A parallel = 171 G) with a partially resolved superhyperfine interaction attributable to at least one directly coordinated nitrogen ligand. Addition of Cu(II) to Gd(III)-alpha-lactalbumin gave an EPR spectrum that was a superposition of signals from the individual Gd(III)- and Cu(II)-alpha-LA spectra. The absence of any magnetic interactions in the Gd(III)-Cu(II)-alpha-lactalbumin species indicated that the two cation sites were more than 10 A apart. On the other hand, addition of Zn(II) to Cu(II)-alpha-lactalbumin gave a set of EPR lines due to free or loosely bound Cu(II), confirming that the Cu(II) was displaced by zinc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature "phase diagrams"

The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a "phase diagram" in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA.     

High-affinity binding of Ca2+ to bovine alpha-lactalbumin in the absence and presence of EGTA.

Literature values for the Kd for Ca2+ in bovine alpha-lactalbumin range over 3 orders of magnitude. There is a difference between two results obtained with EGTA as a metal-ion buffer, partly because different values for the Kd of Ca2+-EGTA were used in the calculations, and a much wider difference between results obtained in the presence and absence of EGTA, which has been attributed to an interaction between EGTA and the protein. Titrations in a flow-dialysis cell showed that Mn2+ competed with Ca2+ for the high-affinity site on the protein, and the results, combined with a Kd for Mn2+ of 2.1 +/- 0.1 microM, which was determined fluorimetrically, gave a Kd for Ca2+ of 1.3 +/- 0.1 nM. When alpha-lactalbumin containing 45Ca2+ was titrated with EGTA in a flow-dialysis cell, and widely accepted metal-chelation data for EGTA were used in the calculations, a Kd for Ca2+ of 1.10 +/- 0.03 nM was obtained. The results from the two methods are so similar as to indicate that the affinity for Ca2+ was unaffected by the presence of EGTA.     

Comparison of the binding of Na+ and Ca2+ to bovine alpha-lactalbumin

alpha-Lactalbumin is a metal-binding protein which binds Ca2+- and Na+-ions competitively to one specific site, giving rise to a large conformational change of the protein. For this reason, the enthalpy change of binding Ca2+ to apo-alpha-lactalbumin (delta Ho) is strongly dependent on the concentration of Na+ ions in the medium. From that relationship a molar enthalpy of -145 +/- 3 kJ X mol-1 is calculated for the Ca2+-binding at pH 7.4 and 25 degrees C, while a delta Ho of -5 +/- 3 kJ X mol-1 is found to substitute a complexed Na+ by a Ca2+-ion. These measurements also allowed us to calculate a binding constant for Na+ of 195 +/- 18 M-1. The molar enthalpy of Na+-loading was found to be -142 +/- 3 kJ X mol-1, a value very close to delta Ho of the binding of Ca2+ to alpha-lactalbumin. Both enthalpy changes in binding Ca2+ and Na+ are independent of the protein concentration. These exothermic values are in agreement with the hypothesis that both Na+- and Ca2+-ions are able to induce the same conformational change in alpha-lactalbumin upon which hydrophobic regions are removed from the solvent, yielding a less hydrophobic protein. The latter is confirmed by means of affinity measurements of the hydrophobic fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulphonate](bis-ANS) to alpha-lactalbumin. The association constant (Ka) decreased from (6.6 +/- 0.5) X 10(4) M-1 in the absence of NaCl to (2.7 +/- 0.2) X 10(4) M-1 in 75 mM NaCl, while the maximum intensity (Imax) of the binary bis-ANS-alpha-lactalbumin complex remained constant at 0.44 +/- 0.02 (arbitrary units). The Ka value of bis-ANS for Ca2+-alpha-lactalbumin was determined at (1.7 +/- 0.2) X 10(4) M-1 and Imax was 0.43 +/- 0.02 (arbitrary units). The difference in hydrophobicity between the two conformational states of the protein was further demonstrated by adsorption experiments of both conformers to phenyl-Sepharose. Apo-alpha-lactalbumin, hydrophobically bound to phenyl-Sepharose, can be eluted by adding Ca2- or Na+-solutions.     

Calorimetric experiments of Mn2+ -binding to alpha-lactalbumin

We measured by batch microcalorimetry the standard enthalpy change delta H degrees of the binding of Mn2+ to apo-bovine alpha-lactalbumin; delta H degrees = -90 +/- k J.mol-1. The binding constants, KMn2+, calculated from the calorimetric and circular dichroism titration curves, are (4.6 +/- 1).10(5) M-1 and (2.1 +/- 0.4).10(5) M-1, respectively. Batch calorimetry confirms the competitive binding Ca2+, Mn2+ and Na+ to the same site. The relatively small enthalpy change for Mn2+ binding compared to Ca2+ binding favours a model of a rigid and almost ideal Ca2+-complexating site, different from the well-known EF-hand structures. Cation binding to the high-affinity site most probably triggers the movement of an alpha-helix which is directly connected to the complexating loop.     

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.     

Identification of regions responsible for the open conformation of S100A10 using chimaeric S100A11-S100A10 proteins

S100A11 is a dimeric EF-hand calcium-binding protein. Calcium binding to S100A11 results in a large conformational change that uncovers a broad hydrophobic surface used to interact with phospholipid-binding proteins (annexins A1 and A2) and facilitate membrane vesiculation events. In contrast with other S100 proteins, S100A10 is unable to bind calcium due to deletion and substitution of calcium-ligating residues. Despite this, calcium-free S100A10 assumes an 'open' conformation that is very similar to S100A11 in its calcium-bound state. To understand how S100A10 is able to adopt an open conformation in the absence of calcium, seven chimaeric proteins were constructed where regions from calcium-binding sites I and II, and helices II-IV in S100A11 were replaced with the corresponding regions of S100A10. The chimaeric proteins having substitutions in calcium-binding site II displayed increased hydrophobic surface exposure as assessed by bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'disulfonic acid, dipotassium salt) fluorescence and phenyl-Sepharose binding in the absence of calcium. This response is similar to that observed for Ca2+-S100A11 and calcium-free S100A10. Further, this substitution resulted in calcium-insensitive binding to annexin A2 for one chimaeric protein. The results indicate that residues within site II are important in stabilizing the open conformation of S100A10 and presentation of its target binding site. In contrast, S100A11 chimaeric proteins with helical substitutions displayed poorer hydrophobic surface exposure and, consequently, unobservable annexin A2 binding. The present study represents a first attempt to systematically understand the molecular basis for the calcium-insensitive open conformation of S100A10.