RESUMO
In order to assess the importance of a variety of environmental factors on the structure of bovine prothrombin fragment 1, we have examined acrylamide quenching of fragment 1 intrinsic fluorescence. Tryptophan exposure, determined from Stern-Volmer plots, is heterogeneous with one or more of the three fragment 1 tryptophans being exposed to solvent. In the presence of Ca2+ or Mg2+ even the most accessible tryptophan(s) are relatively buried. Only small differences in tryptophan exposure may exist between fragment 1-Ca2+ and fragment 1-Mg2+ complexes. Lowering pH, on the other hand, results in increased tryptophan exposure. Finally, structural isomers of fragment 1 which exist in the absence of metal ions have identical tryptophan exposure as determined by acrylamide quenching and fluorescence intensity.
Assuntos
Fragmentos de Peptídeos , Precursores de Proteínas , Protrombina , Triptofano , Acrilamidas , Animais , Cálcio , Bovinos , Concentração de Íons de Hidrogênio , Isomerismo , Cinética , Magnésio , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
The crystal and molecular structure of a dipeptide containing a blocked gamma-carboxyglutamyl (Gla) residue is presented. Two intermolecular hydrogen bonds link the amides with carbonyl groups in the dipeptide backbone, but the protected gamma-carboxy groups on the modified glutamic acid are not hydrogen bonded.
Assuntos
Ácido 1-Carboxiglutâmico , Glutamatos , Ácido 1-Carboxiglutâmico/análogos & derivados , Ácido 1-Carboxiglutâmico/análise , Sequência de Aminoácidos , Fenômenos Químicos , Química , Glutamatos/análogos & derivados , Ligação de Hidrogênio , Modelos MolecularesRESUMO
TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.
Assuntos
Cálcio/farmacologia , Corantes Fluorescentes/metabolismo , Magnésio/farmacologia , Naftalenossulfonatos/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas , Protrombina/metabolismo , Animais , Bovinos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Especificidade da Espécie , Espectrometria de FluorescênciaRESUMO
A kinetic model is derived for the interaction of bovine prothrombin fragment 1 with calcium ions. The model requires binding of a minimum of two calcium ions for induction of the observed biphasic fluorescence decrease as a function of time. The model is shown to be consistent with experimental kinetic and equilibrium data by fitting theoretical curves for the biphasic fluorescence change to the data through exact solution of the nonlinear differential rate equations derived from the model. The rate constants for the binding of these two required calcium ions are calculated from the solutions as best fit parameters. The thermodynamic equilibrium constants, K1 and K2, for the binding of these two calcium ions are calculated from ratios of the forward and reverse rate constants as 0.6 X 10(4) and 5.4 X 10(4), respectively. Thus, the model correctly predicts positively cooperative calcium ion binding for at least the two calcium ions required to induce fluorescence quenching.
Assuntos
Cálcio/metabolismo , Modelos Químicos , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas , Protrombina/metabolismo , Animais , Sítios de Ligação , Bovinos , Cinética , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
Variation of pH strongly affects the fluorescence intensity of human prothrombin fragment-1 in a manner suggesting contributions from a number of protropic equilibria including groups with apparent pKa values near 3.0. These results suggest a structural role for pK1a of gamma-carboxyglutamic acid noieties. Added calcium ions (9 mM calcium chloride) quench the fluorescence titration curve uniformly above pH 4. Below pH 4, however, the titration curve in the presence of calcium ions suggests that calcium-ion-dependent processes leading to fluorescence quenching are pH-dependent. Upon back titration of human fragment-1, from pH 9, hysteresis is observed. Human prothrombin fragment-2 fluorescence titration curves are relatively broad at low pH suggesting the titration of normal carboxyl groups. The titration curves of fragment-2 are not affected by the presence of calcium ions, and hysteresis occurs upon back titration from low pH values. Circular dichroism (CD) Cotton effects appear at 232 nm and 280 nm and a trough appears at 203 nm in the CD spectrum of human prothrombin fragment-2. The Cotton effects in the region from 230 nm to 300 nm are sensitive to pH, ellipticity values at 232 nm increasing from approximately 300 at pH 2.5 to 1300 (degree-cm/decimole) at neutral pH and finally become negative at high pH values. In contrast to fragment-1, at neutral pH the fragment-2 Cotton effect at 232 nm is insensitive to the presence of 8 mM calcium chloride.
Assuntos
Protrombina , Cálcio/farmacologia , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Fenômenos Físicos , Física , Espectrometria de FluorescênciaRESUMO
Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.
Assuntos
Cálcio , Formiatos , Magnésio , Compostos Organofosforados , Simulação por Computador , Modelos Químicos , Estrutura Molecular , Fosfolipídeos , Teoria QuânticaRESUMO
25Mg+2 ion NMR studies of complexes of magnesium ions with acetate and malonate ligands have yielded apparent quadrupolar coupling constants, chi, of approximately 1.5 MHz. The aquo magnesium ion yields a smaller chi value of 0.12 MHz, consistent with its expected higher symmetry. chi values for magnesium ion: acetate and magnesium ion: malonate complexes are utilized to calculate observed linewidths for magnesium ion: bovine prothrombin fragment 1 and magnesium ion: human Factor XII interactions. These calculated values are compared with observed values and implications of the agreement are discussed.
Assuntos
Acetatos , Magnésio , Malonatos , Animais , Bovinos , Fator VIII/metabolismo , Humanos , Cinética , Magnésio/metabolismo , Ligação Proteica , Protrombina/metabolismo , Relação Estrutura-AtividadeRESUMO
The recent suggestion that the apparent cooperativity seen in the binding of Ca(II) ions to prothrombin fragment 1 is due to protein aggregation is evaluated. Since (1) we find that the Ca(II) ion binding is not dependent upon protein concentration, (2) the analytical expression for the equilibrium constant of the aggregation model is unrealistically large when evaluated at realistic Ca(II) ion concentrations, and (3) a very simple allosteric cooperative binding model (Monod) can be shown to fit the experimental data, we conclude that the aggregation explanation for the apparent cooperativity in the Ca(II) ion binding by prothrombin fragment 1 is not correct.
Assuntos
Cálcio/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , Animais , Cátions Bivalentes , Bovinos , Diálise , Substâncias Macromoleculares , Matemática , Modelos Químicos , Fragmentos de Peptídeos/química , Ligação Proteica , Precursores de Proteínas/química , Protrombina/química , TermodinâmicaRESUMO
Human and bovine factor X contain 11 and 12 glutamyl residues, respectively, within the first 40 amino terminal residues that are post-translationally modified to gamma-carboxyglutamyl (Gla) residues. We have measured calcium ion binding to human factor X by equilibrium dialysis. This is the first examination of calcium ion binding to human factor X. We have also re-examined the equilibrium dialysis binding of calcium ions to bovine facor X in order to compare the two species. The data was analysed using a variety of models that allow for more than one class of binding site and for co-operativity among binding sites. Calcium ion binding to human factor X fits a model that had two classes of sites: one class with a single site that had an affinity of 0.1 mM and a second class with 19 equivalent, non-interacting sites with an average affinity of 3.5 mM. There was no evidence for co-operativity in calcium ion binding. Calcium ion binding to bovine factor X was best stimulated by a model that assumed one tight site, four co-operative sites, and 18 equivalent, non-interacting sites. To examine the co-operativity seen in calcium ion binding to bovine factor X, calcium ion binding to isolated Gla region (residues 1-44) and Gla-domainless factor X was measured by equilibrium dialysis. Calcium ion binding to Gla-domainless factor X was simulated by a model that had two classes of sites: one class with a single site that had an affinity of 0.25 mM, and a second class that had 15 sites with very low affinity sites (greater than 15 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Cálcio/metabolismo , Bovinos/sangue , Fator X/metabolismo , Ácido 1-Carboxiglutâmico , Animais , Diálise , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Protrombina/metabolismo , Especificidade da EspécieRESUMO
Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(2+)-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin-fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2 microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol% PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater than or equal to 10 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.
Assuntos
Lipossomos/metabolismo , Microscopia de Fluorescência , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Protrombina/metabolismo , Animais , Cálcio/farmacologia , Bovinos , Fluoresceína , Fluoresceínas , Corantes Fluorescentes , Glicerol/farmacologia , Cinética , Luz , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Espalhamento de Radiação , SoluçõesRESUMO
Circular dichroism spectroscopy was used to investigate the structure of bovine prothrombin fragment 1 (BF1) and related proteins in several environments. The conformational change induced in BF1 by the addition of Mg[II] ions was found to be different from that induced by Ca[II] or Sr[II]. The Ca[II] and Sr[II] conformations appear to differ only slightly from the apo-metal conformation. The conformation of the 1-45 fragment of prothrombin, however, is markedly different than the conformation of the same fragment in the presence of either Ca[II] of Mg[II]; both of the latter structures differ substantially from one another. The presence of phospholipids has almost no effect on the structure of either BF1 or the 1-45 fragment; in the presence of both phospholipids and Ca[II] a structural change is seen for the 1-45 fragment but not BF1 (relative to the protein alone). The addition of phospholipids to the Mg[II]/BF1 structure did not induce a CD-detectable conformational change, while the addition of phospholipids to the Ca[II]/BF1 or Sr[II]/BF1 structures induced a change to a conformation similar in secondary structure composition to the relative apometal structures.
Assuntos
Fragmentos de Peptídeos/síntese química , Precursores de Proteínas/síntese química , Protrombina/síntese química , Animais , Cátions Bivalentes/farmacologia , Bovinos , Dicroísmo Circular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/efeitos dos fármacos , Peptídeos/química , Peptídeos/efeitos dos fármacos , Fosfolipídeos/farmacologia , Conformação Proteica/efeitos dos fármacos , Precursores de Proteínas/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Protrombina/efeitos dos fármacos , Difração de Raios XRESUMO
The determination of binding constants of metal ions to biomolecules is approachable via many techniques. Metal ion NMR spectroscopy is an alternative to more traditional techniques and is complementary to them, particularly in investigations of the interactions of metal ions with relatively small peptides containing multiple ionizing groups. The method requires relatively small amounts of material, is fairly fast, and is carried out at equilibrium. Our study has been of calcium and magnesium ion binding to gamma-carboxyglutamic acid (Gla)-containing peptides. Dissociation constants of approximately 0.6 mM for the binding of either metal ion to Z-D-Gla-D-Gla-OMe have been obtained. The procedure for determination of these constants via metal ion NMR is discussed.
Assuntos
Cálcio , Dipeptídeos , Glutamatos , Magnésio , Cinética , Espectroscopia de Ressonância Magnética , Ligação ProteicaRESUMO
3-SLHis-105-RNase A is an active derivative of ribonuclease A (RNase A) spin-labeled at the 3 position of the imidazole ring of histidine-105. The spin-labeled enzyme has been modified by urea denaturation, reduction, reduction-carboxymethylation, performic acid oxidation, and digestion with proteolytic enzymes in order to monitor changes in the geometry of the protein by changes in the electron paramagnetic resonance (EPR) spectrum of the nitroxide spin-label probe. The results of these experiments indicate that the spin-label attached to histidine-105 of RNase A is sensitive to modifications affecting the conformational integrity of the molecule and to the reconstituting effects of various active-center ligands.
Assuntos
Ribonucleases , Marcadores de Spin , Animais , Soluções Tampão , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Pâncreas/enzimologia , Conformação Proteica , Ribonucleases/metabolismoRESUMO
The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.
Assuntos
Cálcio , Fragmentos de Peptídeos , Protrombina , Animais , Bovinos , Concentração de Íons de Hidrogênio , Ligação Proteica , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
Equilibrium geometries and binding energies of model "salt" or "ion" bridge systems have been computed by ab initio quantum chemistry techniques (GAUSSIAN82) and by empirical force field techniques (AMBER2.0). Formate and dimethyl phosphate served as anions in the model compounds while interacting with several organic cations, including methyl ammonium, methyl guanidinium, and divalent metal ion (either Mg2+ or Ca2+) without and with an additional chloride; and a divalent metal ion (either Mg2+ or Ca2+), chloride, and four water molecules of hydration about the metal ion. The majority of the quantum chemical computations were performed using a split-valence basis set. For the model compounds studied we find that the ab initio optimized geometries are in remarkably good agreement with the molecular mechanics geometries. Several calculations were also performed using diffuse fractions. The formate anion binds these model cations more strongly than does dimethyl phosphate, while the organic cation methyl ammonium binds model anions more strongly than does methyl guanidinium. Finally, in model compounds including organic anions, Mg2+ or Ca2+ and four molecules of water, and a chloride anion, we find that the equilibrium structure of the magnesium complex involves a solvent separated ion pair (the magnesium ion is six coordinate), whereas the calcium ion complex remains seven coordinate. Molecular mechanics overestimates binding energies, but the estimates may be close enough to actual binding energies to give useful insight into the details of salt bridges in biological systems.
Assuntos
Físico-Química , Modelos Moleculares , Fenômenos Químicos , Simulação por Computador , ÍonsRESUMO
Calcium-mediated binding of the radioiodinated peptide representing residues 1-45 of bovine prothrombin to single bilayer phospholipid vesicles composed of phosphatidylserine from bovine brain and synthetic 1-palmitoyl-2-oleoyl-phosphatidylcholine (25:75 PS/PC) has been studied over peptide concentrations from 0.33 microM to 3.75 microM and at a calcium concentration of 1.0 mM. The binding isotherm for the interaction between the radioiodinated peptide and PS/PC vesicles fits a model in which there is noncooperative binding of the peptide to non-interacting sites on the phospholipid bilayer. A dissociation constant determined at these conditions is 11.8 microM compared to 1.0 microM for prothrombin fragment 1.
Assuntos
Ácido 1-Carboxiglutâmico/metabolismo , Cálcio/fisiologia , Lipossomos , Protrombina/metabolismo , Animais , Bovinos , Cinética , Substâncias Macromoleculares , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Relação Estrutura-Atividade , TermodinâmicaRESUMO
An equilibrium dialysis technique for examining magnesium binding to macromolecules is described. The technique is used to determine the binding constants of magnesium to human prothrombin. This procedure should be of great utility for many biochemical systems which exhibit magnesium affinity.
Assuntos
Diálise/métodos , Magnésio/metabolismo , Protrombina/metabolismo , Humanos , Técnicas In Vitro , Cinética , Ligação ProteicaRESUMO
A unique method is presented for the determination of the critical number of ligands that must bind to a macromolecule to elicit a spectroscopic response. This method is based on analysis of ligand binding data. For example, four Ca2+ and two Mg2+ ions are necessary for mirroring the relative decrease in the intrinsic fluorescence of bovine prothrombin fragment 1. For application of the method, ligand loading and relative spectroscopic response data must be measured over a full range of concentrations.