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.     

Structural characteristics of protein binding sites for calcium and lanthanide ions

Surveys of X-ray structures of Ca2+-containing and lanthanide ion-containing proteins and coordination complexes have been performed and structural features of the metal binding sites compared. A total of 515 structures of Ca2+-containing proteins were considered, although the final data set contained only 44 structures and 60 Ca2+ binding sites with a total of 323 ligands. Eighteen protein structures containing lanthanide ions were considered with a final data set containing eight structures and 11 metal binding sites. Structural features analysed include coordination numbers of the metal ions, the identity of their ligands, the denticity of carboxylate ligands, and the type of secondary structure from which the ligands are derived. Three general types of calcium binding site were identified in the final data set: class I sites supply the Ca2+ ligands from a continuous short sequence of amino acids; class II sites have one ligand supplied by a part of the amino acid sequence far removed from the main binding sequence; and class III sites are created by amino acids remote from one another in the sequence. The abundant EF-hand type of Ca2+ binding site was under-represented in the data set of structures analysed as far as its biological distribution is concerned, but was adequately represented for the chemical survey undertaken. A turn or loop structure was found to provide the bulk of the ligands to Ca2+, but helix and sheet secondary structures are slightly better providers of bidentate carboxylate ligation than turn or loop structures. The average coordination number for Ca2+ was 6.0, though for EF-hand sites it is 7. The average coordination number of a lanthanide ion in an intrinsic protein Ca2+ site was 7.2, but for the adventitious sites was only 4.4. A survey of the Cambridge Structural Database showed there are small-molecule lanthanide complexes with low coordination numbers but it is likely that water molecules, which do not appear in the electron density maps, are present for some lanthanide sites in proteins. A detailed comparison of the well-defined Ca2+ and lanthanide ion binding sites suggests that a reduction of hydrogen bonding associated with the ligating residues of the binding sites containing lanthanide ions may be a response to the additional positive charge of the lanthanide ion. Major structural differences between Ca2+ binding sites with weak and strong binding affinities were not obvious, a consequence of long-range electrostatic interactions and metal ion-induced protein conformational changes modulating affinities.     

Amiodarone-liposome interaction: a multinuclear NMR and X-ray diffraction study

Amiodarone, a potent antiarrhythmic drug, is widely used in cardiology. Its electrophysiological effects, as well as many of its side effects, seem to involve lipids. We report here a multinuclear NMR and X-ray diffraction study of amiodarone in egg phosphatidylcholine liposomes and lipid multilayers. In proton NMR experiments, amiodarone alters the signal from the lipid trimethyl ammonium group for pH values ranging from 3.2 to 8.4; cholesterol does not cause this alteration. The addition of SCN- changes both the proton and phosphorus NMR spectra of liposomes containing amiodarone. For both proton and carbon NMR, amiodarone modifies the signal from the lipid methylene groups, but to a far lesser extent than does cholesterol. Incorporation of amiodarone in EPC bilayers also modifies the low-angle X-ray diffraction patterns, decreasing the lamellar repeat period at low water contents, but swelling the fluid spaces between bilayers at high water contents. Electron density profiles and modeling studies using the X-ray data indicate that amiodarone decreases the bilayer thickness and adds electron density at the interfacial region of the bilayer. Our analysis of the NMR and X-ray data indicates that the iodine atoms of amiodarone are located near the hydrocarbon/water interface and that the tertiary amine of amiodarone is in the headgroup region of the bilayer.     

Analysis of a calcium ion binding system composed of two different sites

Using murexide (Mx), a metallochromic indicator, and a dual wavelength spectrophotometer with a high signal-to-noise ratio, the Ca⁺⁺ binding in a system containing two classes of binding sites was studied. Solutions with solute containing one or two classes of Ca⁺⁺ binding sites and without such solute were titrated with Ca⁺⁺ using Mx as an indicator of free Ca⁺⁺ concentration. Since curvilinear Scatchard plots are obtained from titration curves of solutes containing two classes of binding sites, a computer program was developed to resolve such plots into two linear partial plots, each corresponding to a single class of binding site. The validity of the procedure was examined with solutions of ethylene glycol bis(β-aminoethyl)-N-N′-tetraacetic acid, adenosine triphosphate (EGTA, ATP), or a mixture thereof. The method was also applied to biological material and it was found that a protein fraction isolated from rat skeletal muscle sarcotubular membranes, termed Fraction-2 (Fr-2), has two classes of binding sites for Ca⁺⁺; the association constants of the high affinity site and low affinity site are 4.3 × 10⁵ M^-1 and 9 × 10³ M^-1, respectively. The advantages and limitations of this methodology are discussed.     

Interaction of vanadate with monosaccharides and nucleosides: a multinuclear NMR study

Proton, 13C and 51V nuclear magnetic resonance spectroscopy has been used to study the interaction of vanadate with several molecules containing more than one hydroxyl group, including various aldoses and nucleosides. The aldoses D-mannose and D-ribose mainly form tridentate complexes, of trigonal bipyramidal geometry, with vanadate at pH 7. These sugars use three consecutive hydroxyl groups, cis to each other, of their pyranose forms to bind vanadate in those cyclic triesters. Other aldoses, like D-glucose, which do not have this unique structural characteristic, do not form tridentate complexes, but can form weaker bidentate cyclic diesters using two consecutive pyranose cis hydroxyl groups. Of course, the pyranose forms of D-mannose and D-ribose, as well as the furanose form of D-ribose, also yield cyclic diesters of vanadate. All these aldoses form weak monodentate noncyclic monoesters of tetrahedral geometry using a single hydroxyl group. The nucleosides uridine, cytidine and adenosine form two complexes of trigonal bipyramidal geometry with vanadate. In these complexes, having 1:1 and 2:1 ligand-to-metal stoichiometries, the nucleosides form cyclic diesters with vanadate using their C2, and C3, hydroxyl groups.     

Instability of purified 9-kDa porcine intestinal calcium-binding protein in the absence of calcium

Purified 9-kDa porcine intestinal calcium-binding protein (ICaBP, Calbindin D9K) is unstable when stored at 4 degrees C in the absence of Ca(II). Cleavage of the polypeptide occurs producing approximately 5.2- and 3.7-kDa fragments. The former dimerizes giving a species which migrates on sodium dodecylsulfate-urea gels with an Mr 13,700, in contrast to the observed Mr 11,000 for native ICaBP. The fragmentation also results in an irreversible loss of high affinity Ca(II) bound to ICaBP prevents fragmentation. The dimer can be isolated from aged preparations of apo-ICaBP and is stable on further storage with or without Ca(II) present. The observed molecular weights of the fragments along with the amino acid analysis and ultraviolet spectra of the dimer suggest cleavage of the polypeptide chain of ICaBP in the vicinity of residue 49.     

A comparative study of the backbone dynamics of two closely related lipid binding proteins: Bovine heart fatty acid binding protein and porcine ileal lipid binding protein

The backbone dynamics of bovine heart fatty acid binding protein (H-FABP) and porcine ileal lipid binding protein (ILBP) were studied by 15N NMR relaxation (T1 and T2) and steady state heteronuclear 15N{1H} NOE measurements. The microdynamic parameters characterizing the backbone mobility were determined using the model-free approach. For H-FABP, the non-terminal backbone amide groups display a rather compact protein structure of low flexibility. In contrast, for ILBP an increased number of backbone amide groups display unusually high internal mobility. Furthermore, the data indicate a higher degree of conformational exchange processes in the sec-msec time range for ILBP compared to H-FABP. These results suggest significant differences in the conformational stability for these two structurally highly homologous members of the fatty acid binding protein family.     

Different conformation changes induced by calcium and terbium of the porcine intestinal calcium-binding protein

The fluorescence of 1-anilino-8-naphthalenesulfonate (ANS) is progressively enhanced with increasing concentration of it, showing a proportionate blue shift of the emission maximum, by the interaction with the porcine intestinal Ca2+-binding protein (CaBP) in the absence of Ca2+. The apo-CaBP has a single binding site for ANS as determined by the fluorescence change, the apparent dissociation constant (Kd) estimated at 49.1 microM. Addition of Ca2+ or Tb3+ to the ANS-apo-CaBP system is capable of enhancing its fluorescence up to about 2- or 5-fold, respectively, causing further blue shift of the emission maximum. These metal ions do not affect the capacity of ANS binding, but Ca2+ slightly increases the Kd value. Increase of the fluorescence of the ANS-CaBP complex by increasing binding of Ca2+ to it was monophasic, while that with Tb3+ was biphasic, both saturated at the same molar ratio, 2, of added cations to the complex. Biphasic change of response has also been observed in UV absorption of the CaBP with increasing concentration of Tb3+. With a half-saturating concentration of Tb3+, Ca2+ can induce a much higher enhancement of the ANS fluorescence than excess Ca2+ alone. All these results indicate that the CaBP molecule contains a single ANS binding site and the conformation and/or microenvironment surrounding bound ANS of the protein is altered reversibly with binding of Ca2+ or Tb3+ to it and that there are differences between Ca2+- and Tb3+-induced conformation changes around the ANS-binding site and the tyrosine residue of it.     

Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study.

The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human complement protein C9 is a calcium binding protein. Structural and functional implications

Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca2+/mol of C9 with a dissociation constant of 3 micron as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element or domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although we do not know yet the precise structural and functional role of the bound calcium, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.     

A multinuclear NMR study of 2,3-bisphosphoglycerate metabolism in the human erythrocyte

Resonances from 13C, 31P and 1H have been detected simultaneously in suspensions of human erythrocytes using a modified NMR spectrometer equipped with a probe tuned to four different frequencies. The utility of multinuclear NMR in the study of cellular metabolism is demonstrated with an investigation of 13C label flux through the 2,3-bisphosphoglycerate bypass in human erythrocytes. In a single experiment, the respective contributions of this bypass and the pentose-phosphate shunt were found to be 27 and 10% of the total glycolytic rate.     

Structural characterization of the calcium binding protein s100 from adipose tissue

A partial amino acid sequence for bovine adipose tissue S100 was elucidated by characterization of peptides generated by cyanogen bromide cleavage. The cyanogen bromide peptides were aligned by homology with the bovine brain S100 beta sequence. The results demonstrate that adipose S100 beta is probably identical to brain S100 beta, and suggest that S100 beta is a conserved protein among tissues of the same species.