Binding Of gD Research Articles

Stereochemistry of Gd 3+ and Mn 2+ interactions with D-gluconamide derivatives by 13C NMR spectroscopy

Natural abundance 13C nuclear magnetic resonance spectroscopy ( 13C NMR) was used to study the mode of binding of Gd 3+ and Mn 2+ to the polyol portion of several synthetic D-gluconamides. The results indicate that Gd 3+ forms a single, unique binding structure requiring three oxygen atoms. The binding of Mn 2+ to the polyol portion of these compounds appears to be nonspecific. The carbohydrate containing model compounds studied may be used to design new metal-ion chelating agents.

13C-n.m.r.-spectral study of the mode of interaction of Gd 3+ and Mn 2+ with two vicinally di- O- d-galactosylated tripeptides

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd 3+ and Mn 2+ to two vicinally di- O-α- and -β- d-galactosylated tripeptides composed of Gly and l-Thr. Gd 3+ and Mn 2+ appear to interact with the α- d-Gal groups of the di- O-α- d-galactosylated tripeptide at two sites: near O-6′, and in the vicinity of O-2′ and Thr O-3. The metal-ion-binding to the β- d-Gal groups of the di- O-β- d-galactosylated tripeptide indicates that a strong binding-site exists near O-6′ and, possibly, several weak ones near O-3′ and Thr O-3. In the case of the di- O-α- d-galactosylated tripeptides, vicinal glycosylation appears to have little effect on the metal-ion-binding of the α- d-Gal groups.

13C-n.m.r.-spectral study of the mode of binding of Gd 3+ to various glycopeptides

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the mode of binding of Gd 3+ to mono- O-glycosylated l-serine and tripeptides variously composed of Gly and l-Thr. When the amino and carboxyl groups of the amino acid are not blocked, strong interaction of Gd 3+ with them is observed; this is also readily apparent with some related, nonglycosylated peptides. When the amino and carboxyl groups of the amino acid are blocked, noticeable interaction of Gd 3+ with the glycosidic oxygen atom (O-3) and O-2′ for the glycopeptide containing α- d-Gal p, and with O-3 and N-2′ for the glycopeptide containing α- d-Gal pNAc, is observed. Weak interactions are also possible with O-4′ and O-6′ of the glycosyl groups. Although the amino acids were protected, these metal ion-carbohydrate interactions may still be mediated, to some extent, by the acetyl protecting the amino group and by the ester group on the amino acid.

13C-n.m.r.-spectral study of the binding of Gd 3+ to glycophorin

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd 3+ to glycophorin, and also to the tetrasaccharides isolated from glycophorin after treatment of the glycoprotein with NaOH-NaBH 4. Gd 3+ binds to the tetrasaccharide (both in the isolated, reduced form and when still attached to the native glycoprotein), and, especially, to the α-NeuAc residues. In order to cause severe line-broadening of the 13C resonances of α-NeuAc, the ratios of the α-NeuAc residues of glycophorin, and of the isolated, reduced tetrasaccharide, to Gd 3+ were much higher than that needed for causing similar broadening for 2- O-methyl-α-NeuAc-Gd 3+ solutions. These results indicate that the other carbohydrate residues of the tetrasaccharide may be involved in the binding of Gd 3+, producing a stronger metal-ion-binding effect.

Conformational analysis of l-alanyl- l-tryptophan and glycyl- l-histidine from nuclear-nuclear and electron-nuclear spin relaxation

The conformations and dynamical behavior of alanyl-tryptophan (AW) and glycylhistidine (GH) in aqueous solutions were investigated by 1H and 13C nuclear magnetic resonance and relaxation, some experiments being performed in the presence of the Gd 3+ ion. The 13C relaxation in diamagnetic solutions shows that the rates of internal motions of these dipeptides are slow compared to those of the overall re-orientational motions of these molecules, except for the alanine methyl and glycine methylene group in AW and GH, respectively. The main conformations of the dipeptides were determined by means of the 1H and 13C longitudinal relaxation enhanced by Gd 3+ bound selectively to the carboxylate residue, forming (AW) 2Gd and (GH) 2Gd complexes. These experiments show in particular that the G′ rotamer about the tryptophan or histidine C α-C β bond ( χ 1 = 180°) is the most populated one with P G′ ≅ 0.7, whereas the angle χ 2 defining the orientations of the indole or imidazole residues about C β- C γ is independent of χ 1. 3 J HH and proton-proton longitudinal relaxation measurements in free AW and GH show that the binding of Gd 3+ does not induce appreciable changes in the conformations of the dipeptides and allows unambiguous determination of the signs of the torsional angles χ 2 and φ, the latter defining the relative orientations of the two amino acid residues.

13C-N.M.R.-Spectral study of the mode of binding of Mn 2+ and Gd 3+ to N-acetyl-α-neuraminic acid

Natural-abundance, 13C-n.m.r. spectroscopy was used to study the binding of Gd 3+ and Mn 2+ to N-acetyl-2- O-methyl-α-neuraminic acid (2) and to methyl N-acetyl-2- O-methyl-α-neuraminate (3). The results showed that Gd 3+ and Mn 2+ bind in the region of the glycerol-1-yl side-chain and the 5-acetamido group of compound 3. When the α-NeuAc derivative contains a carboxylate anion, as in compound 2, multiple, metal-ion-binding sites occur, involving the head (the carboxyl end) and the tail (the glycerol-1-yl and 5-acetamido groups) of the molecule.

Crystal structures of hen egg-white lysozyme complexes with gadolinium(III) and gadolinium(III)-N-acetyl-D-glucosamine.

Analysis at 0.25 nm resolution of the crystal structures of lysozyme-Gd(III) and lysozyme-Gd(III)-N-acetyl-D-glucosamine (GlcNac), prepared by diffusion methods, show that there are two main binding positions for Gd(III), one of which is close to glutamic acid-35 and the other close to aspartic acid-52. The two sites are 0.36 nm part. There is no evidence for the weak binding of Gd(III) to any of the eight other carboxy groups of lysozyme. In the presence of Gd(III), the binding of GlcNac is similar to that observed for the binding of the beta-anomer in subsite C. There are numerous small conformational changes in the protein on binding (Gd(III) and the sugar, and these have been quantified to a first approximation by real-space refinement. These changes are similar in both structures, and involve, among other small movements, shifts of one of the disulphide bridges by up to 0.05 nm. The movement of residues 70--74 observed in the binary complex of lysozyme-GlcNac [Perkins, Johnson, Machin & Phillips (1978) Biochem. J. 173-617] is not observed in the ternary complex of lysozyme-Gd(III)-GlcNac. The nature of the lysozyme-Gd(III) complex is discussed in the light of evidence from other crystallographic studies and n.m.r. solution studies. Preliminary findings for a lysozyme-Gd(III) complex prepared by co-crystallization methods are reported.

The gross architecture of an antibody-combining site as determined by spin-label mapping

1. A series of Dnp (dinitrophenyl) nitroxide spin labels was used to map the dimensions of the combining site of the Dnp-binding immunoglobulin A myeloma protein MOPC 315. The method compares the observed e.s.r. (electron-spin-resonance) hyperfine splittings with those calculated on the basis of different postulated motions for the spin label. The analysis is complicated by the sensitivity of the e.s.r. hyperfine splitting to the overall ;tumbling' time of the antibody-hapten complex and the polarity of the spin-label's environment. When these effects are considered quantitatively, it is then possible to determine the degree of mobility of each hapten which is allowed by the shape of the combining site. 2. The dinitrophenyl ring is rigidly held, and the depth of the site is 1.1-1.2nm and has lateral dimensions at the entrance to the site >/=0.6nmx0.9nm. The analysis of the results for spin-labelled haptens with chiral centres allows these lateral dimensions to be refined to 0.8nm and 1.1nm, and it is shown that the site is asymmetric with respect to the plane of the dinitrophenyl ring. 3. A polarity profile of the combining site was also obtained and a positively charged amino acid residue, possibly arginine-95(L) (light chain), was located at the entrance to the site. 4. The binding of Gd(III) to the antibody-hapten complexes results in quenching of the e.s.r. signal of the nitroxide. By using La(III) as a control, the paramagnetic contribution to the quenching is measured. 5. Analysis of the differential quenchings of the enantiomers of two five-membered nitroxide ring spin labels gives two possible locations of the metal-binding site. One of these is equidistant (0.7nm) from each of the three dinitrophenyl aromatic protons, and nuclear-magnetic-resonance relaxation studies, at 270MHz, on solutions of dinitrobenzene, Gd(III) and the Fv fragment (variable region of heavy and light chain) from protein MOPC 315 support this location for the metal site. 6. The e.s.r. and metal-binding data were then compared with the results of a model of the combining site constructed on the basis of framework invariance in immunoglobulins [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol.41, in the press]. The overall agreement is very good. Assignments of possible chelating groups for the metal can be made.

Lanthanide binding to cardiac and skeletal muscle microsomes: Effects of adenosine triphosphate, cations, and ionophores

Lanthanide (gadolinium, Gd) binding to cardiac and skeletal muscle microsomes was studied, and high- and low-affinity sites were identified. The high-affinity constant was 10 6 M −1, and there were 131 and 107 nmol/mg bound to this site in dog heart and rabbit skeletal muscle, respectively. Zn 2+, Cd 2+, Al 3+, and Ca 2+ (5 m m) inhibited binding, especially of the high-affinity site. Ionophores X537A (10 μ m) and A23187 (1–2 μ m) increased lanthanide binding and did not cause release. Addition of ATP in low concentration (20–50 μ m) increased the binding of Gd without hydrolysis of the ATP. The extra binding induced by ATP was blocked by heating the microsomes and was reversed by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. High concentrations (10 −4–10 −3, m) of ATP blocked extra Gd binding by competitive chelation. The Ca 2+-activated ATPase was inhibited by Gd and stimulated by X537A. The Gd did not block the ionophore-stimulated increase in Ca 2+-ATPase activity. It is postulated that lanthanides bind predominantly to the ionophoric component of the Ca-transport site rather than the hydrolytic site and that ATP may facilitate such binding without being split.

The use of gadolinium as a probe in the Fc region of a homogeneous anti-(type-III pneumococcal polysaccharide) antibody.

The binding of gadolinium [Gd(III)] to a homogeneous rabbit anti-(type-III pneumococcal polysaccharide) IgG (immunoglobulin G) and its Fab (N-terminal half of heavy and light chain) and Fc (C-terminal half of heavy-chain dimer) fragments was demonstrated by measurements of solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 the binding of Gd(III) to the Fc fragment is much tighter (KD approx. 5 micronM) than binding to the Fab fragment (KD approx. 250 micronM). The binding of Gd(III) to the whole IgG molecule (KD approx. 4 micronM) is very similar to that for the Fc fragment alone. This specificity of binding to the Fc region allows the use of Gd(III) as a probe of the Fc conformation. The environment of the Gd(III) in the Fc region of whole IgG is not affected by the presence of octasaccharide derived by hydrolysis of type-III pneumococcal polysaccharide, but the corresponding 28-unit saccharide does cause detectable changes. The addition of 16-unit saccharide to anti-(SIII polysaccharide) IgG in the presence of Gd(III) does not change the solvent water proton relaxation rate, although aggregation does occur. The effects of the 28-unit saccharide may be explained therefore by a change in the tumbling time of the IgG. From a study of the effect of various antigen/antibody ratios, it is concluded that the 28-unit-saccharide-induced changes in the Gd(III) environment in the Fc region are caused by the specific geometrical structure of the antigen-antibody complexes formed, and not simply by occupancy of the combining sites on the antibody.

Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315.

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment...

The binding of lanthanides to non-immune rabbit immunoglobulin G and its fragments

The binding of Gd(III) to rabbit IgG (immunoglobulin G) and the Fab (N-terminal half of heavy and light chain), (Bab')2 (N-terminal half of heavy and light chains joined by inter-chain disulphide bond), Fc (C-terminal half of heavy-chain dimer)and pFc' (C-terminal quarter of heavy-chain dimer) fragments was demonstrated by measurements of the enhancement of the solvent-water proton relaxation rates in the appropriate Gd(III) solutions. At pH 5.5 there are six specific Gd(III)-binding sites on the IgG. These six sites can be divided into two classes; two very 'tight' sites on the Fc fragment (Kd approx. 5 muM) and two weaker sites on each Fab region (Kd approx. 140 muM). Ca(II) does not apparently compete for these metal-binding sites. The metal-binding parameters for IgG can be explained as the sum of the metal binding to the isolated Fab and Fc fragments, suggesting that there is no apparent interaction between the Fab and Fc regions in the IgG molecule. The binding of Gd(III) to Fab and Fc fragments was also monitored by measuring changes in the electron-spin-resonance spectrum of Gd(III) in the presence of each fragment and also by monitoring the effects of Gd(III) on the protein fluorescence at 340 nm (excitation 295 nm). The fluorescence of Tb(III) solutions of 545 nm (excitation 295 nm) is enhanced slightly on addition of Fab or Fc.

Interaction of lanthanide ions with bovine factor X and their use in the affinity chromatography of the venom coagulant protein of Vipera russelli

The substitution of trivalent lanthanide ions for Ca(II) in the Ca(II)-DEPENDENT ACTIVATION OF BOVINE Factor X by the coagulant protein of Russell's viper venom was studied at pH 6.8. Factor X contains two high affinity metal binding sites which bind Gd(III), Sm(III), and Yb(III) with a Kd of about 4 X 10-7 M and four to six lower affinity metal binding sites which bind Gd(III), Sm(III) with a Kd of about 1.5 X 10-5M. In comparison, 1 mol of Factor X binds 2 mol of Ca(II) with a Kd of 3 X 10-4M and weakly binds many additional Ca(II) ions. No binding of Gd(III) to the venom protein was observed. Dy(III), Yb(III), Tb(III), Gd(III), Eu(III), La(III), AND Nd(III) cannot substitute for Ca(II) in the Ca(II)-dependent activation of Factor X by the venom protein at pH 6.8. Kinetic data consistent with the models of competitive inhibition of Ca(II) by Nd(III) yielded a Ki of 1 to 4 X 10-6M. The substitution of lanthanide ions for Ca(II) to promote protein complex formation of Factor X-metal-venom protein without the activation of Factor X facilitated the purification of the coagulant protein from crude venom by affinity chromatography. Using a column containing Factor X covalently bound to agarose which was equilibrated in 10 mM Nd(III), Tb(III), Gd(III), or La(III), the coagulant protein was purified 10-fold in 40% yield from crude venom and migrated as a single band on gel electrophoresis in sodium dodecyl sulfate. These data suggest that lanthanide ions complete with Ca(II) for the metal binding sites of Factor X and facilitate the formation of a nonproductive ternary complex of venom protein-Factor X-metal. Tb(III) fluorescence, with emission maxima at 490 and 545 nm, is enhanced 10,000-fold in the presence of Factor X. The study of the participation of an energy donor intrinsic to Factor X in energy transfer to Tb(III) may be useful in the characterization of the metal binding sites of Factor X.

Quantitative determination of the conformation of cyclic 3′,5′-adenosine monophosphate in solution using lanthanide ions as nuclear magnetic resonance probes

The conformation of cyclic 3′,5′-adenosine monophosphate in deuterium oxide has been determined at pH 2.0 and pH 5.5, using lanthanide ions as paramagnetic nuclear magnetic resonance probes. The lanthanide ion-induced shifts in the nuclear magnetic resonance energy for a given nucleus are dependent on the geometric position of that nucleus relative to the bound lanthanide ion. As expected, these shifts are pseudocontact in origin and are consistent with axial symmetry. Analysis of the concentration dependence of the shift shows that the lanthanide ion is bound to the phosphate entity giving a 1:1 complex. Further, base stacking and other intermolecular interactions are negligible. To confirm the conformation, which is found from a computer search with the above shift data, we have measured the changes in relaxation times, T 1 and T 2, induced by binding of Gd 3+. The geometric dependence of these relaxation effects is different from that of shifts, being dependent only on distance. The agreement of these data with the computer “shift” conformation is satisfactory. Some 31P nuclear magnetic resonance experiments were done to confirm the metal co-ordination position although, here, there are contact contributions to both shift and relaxation. The computer program finds the conformations that have the correct geometry to account for the shift data, by searching all possible conformations. Non-bond rotations were used as a method of changing the pucker of the phosphate and ribose rings, the position of the base being defined by a single bond rotation. The nuclear magnetic resonance data and minimum van der Waals' distances were used as “active filters” in the computer search. At both values of the pH we have found closely related families of solutions, with the pucker of the phosphate and ribose rings roughly similar to those in an approximate X-ray study of cyclic AMP. The orientation of the base varies with pH.

The Interaction of the Lanthanide Ions with Staphylococcal Nuclease

The metal ion-binding site of staphylococcal nuclease has been studied at pH 7.0 by using several lanthanide ions. The binding of Gd(III) or Nd(III) to nuclease was associated with tyrosine perturbations in the ultraviolet spectrum which were shown to include tyrosine-85 and tyrosine-115 by using mononitrotyrosine nuclease derivatives. A binding constant of 9 µ m was estimated by spectrophotometric titration of nitrotyrosine-115-nuclease with Gd(III). The Ca(II) required for the binding of nuclease to deoxythymidine 3′,5′-diphosphate-Sepharose could be replaced by Nd(III). Nd(III) stabilized liganded and unliganded nuclease to trypsin digestion. None of the lanthanide ions replaced Ca(II) in the generation of nuclease activity toward DNA under the conditions employed. Kinetic data compatible with a model of competitive inhibition of Ca(II) by La(III), Pr(III), Nd(III), Gd(III), Dy(III), and Yb(III) yielded inhibition constants of 1 to 2 µ m . These data suggest that the lanthanide ions bind tightly to the Ca(II) binding site of nuclease in the presence or absence of substrate or substrate analogue. As does Ca(II), these ions also enhance the binding of substrate analogue to nuclease.