RESUMO
The mobility of betaCH2 moieties in oxidized and reduced cytochrome b5 was studied by analyzing the 13C relaxation of the J-split components, in terms of C-H dipole-C-H dipole cross correlation rates. A 2D 13C-1H experiment is proposed to measure these rates that provide the internal effective reorientation correlation time for each CH2 moiety. It is found that higher mobility is present in the alpha helices forming the heme pocket. On the contrary, the beta strands, which form the hydrophobic core of the molecule, have the lowest mobility. The general pattern is the same for the oxidized and reduced species, indicating that any oxidation-dependent property detected for backbone NH moieties does not affect the CH2 mobility.
Assuntos
Proteínas de Bactérias/química , Citocromos b5/química , Ressonância Magnética Nuclear Biomolecular , Escherichia coli/enzimologia , Heme/química , Modelos Moleculares , Movimento (Física) , Oxirredução , Conformação ProteicaRESUMO
The Ca2 calbindin protein in which one calcium has been substituted with Ce(III), Yb(III) and Dy(III) displays substantial alignment in high magnetic fields due to the high anisotropy of the metal magnetic susceptibility. This property has allowed the measurement of residual dipolar coupling contributions to 1J(HN) and 2J(HH) couplings of asparagine and glutamine NH2 moieties. Such data have been used to aid structural characterization of these groups. The exploitation of auto-orientation of magnetic anisotropic metalloproteins represents a step ahead in the investigation of the conformational space of peripheral residues that are not fixed by the protein folding.
Assuntos
Metais Terras Raras , Ressonância Magnética Nuclear Biomolecular/métodos , Anisotropia , Asparagina/química , Sítios de Ligação , Calbindinas , Cério/química , Cério/metabolismo , Disprósio/química , Disprósio/metabolismo , Glutamina/química , Magnetismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Metais Terras Raras/química , Metais Terras Raras/metabolismo , Isótopos de Nitrogênio , Estrutura Terciária de Proteína , Proteína G de Ligação ao Cálcio S100/química , Proteína G de Ligação ao Cálcio S100/metabolismo , Itérbio/química , Itérbio/metabolismoRESUMO
Cytochrome b5 in solution has two isomers (A and B) differing by a 180 degrees rotation of the protoporphyrin IX plane around the axis defined by the alpha and gamma meso protons. Homonuclear and heteronuclear NMR spectroscopy has been employed in order to solve the solution structure of the minor (B) form of the oxidized state of the protein and to probe its backbone dynamics in the microsecond--ms timescale in both oxidation states. A family of 40 conformers has been obtained using 1302 meaningful NOEs and 220 pseudocontact shifts and is characterized by high quality and good resolution (rmsd to the mean structure of 0.055 +/- 0.009 nm and 0.103 +/- 0.011 nm for backbone and heavy atoms, respectively). Extensive comparisons of the structural and dynamics changes associated with the A-to-B form interconversion for both oxidation states were subsequently performed. Propionate 6 experiences a redox-state-dependent reorientation as does propionate 7 in the A form. Significant insights are obtained into the role of the protein frame for efficient biological function and backbone mobility is proposed to be one of the factors that could control the reduction potential of the heme.
Assuntos
Citocromos b5/química , Microssomos/enzimologia , Animais , Isomerismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oxirredução , Ratos , Soluções , Relação Estrutura-AtividadeRESUMO
A new experiment, the forward directed quantitative gamma-HCCH-TOCSY for the measurement of the conformation of the five-membered ribosyl unit in RNA oligonucleotides, is presented. The experiment relies on quantification of cross peak intensities caused by evolution of CH, CH-dipole-dipole cross correlated relaxation in non-evolution periods and the resolution enhancement obtainable in forward directed HCC-TOCSY transfer. Cross correlated relaxation rates are interpreted to reveal the sugar conformation of 22 out of 25 nucleotides in an isotopically labelled 25-mer RNA. The results obtained with this new method are in agreement with the conformational analysis derived from 3J(H,H) coupling constants.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Oligorribonucleotídeos/química , Configuração de Carboidratos , Modelos Teóricos , Ressonância Magnética Nuclear Biomolecular/instrumentação , Conformação de Ácido Nucleico , SoluçõesRESUMO
Rotating frame 15N relaxation NMR experiments have been performed to study the local mobility of the oxidized and reduced forms of rat microsomal cytochrome b5, in the microsecond to millisecond time range. Measurements of rotating frame relaxation rates (R1rho) were performed as a function of the effective magnetic field amplitude by using off-resonance radio frequency irradiation. Detailed analysis of the two data sets resulted in the identification of slow motions along the backbone nitrogens for both oxidation states of the protein. The local mobility of reduced and oxidized cytochrome b5 turned out to be significantly different; 28 backbone nitrogens of the oxidized form were shown to participate in a conformational exchange process, while this number dropped to 12 in the reduced form. The correlation time, tauex, for the exchange processes could be determined for 21 and 9 backbone nitrogens for oxidized and reduced cytochrome b5, respectively, with their values ranging between 70 and 280 microseconds. The direct experimental evidence provided in this study for the larger mobility of the oxidized form of the protein is consistent with the different backbone NH solvent exchangeability recently documented for the two oxidation states [Arnesano, F., et al. (1998) Biochemistry 37, 173-184]. Our experimental observations may have significant biological implications. The differential local mobility between the two oxidation states is proposed to be an important factor controlling the molecular recognition processes in which cytochrome b5 is involved.
Assuntos
Citocromos b5/química , Citocromos b5/metabolismo , Microssomos Hepáticos/enzimologia , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Termodinâmica , Animais , Citocromos b5/fisiologia , Transporte de Elétrons , Modelos Moleculares , Isótopos de Nitrogênio , Oxirredução , RatosRESUMO
Rotating-frame 15N relaxation rate (R1 rho) NMR experiments have been performed in order to study the dynamic behavior of the reduced recombinant high-potential iron-sulfur protein iso I (HiPIP I) from Ectothiorhodospira halophila, in the microsecond to ms time range. Measurements of R1 rho were performed as a function of the effective spinlock magnetic field amplitude by using both on and off-resonance radio frequency irradiation. The two data sets provided consistent results and were fit globally in order to identify possible exchange processes in an external loop of the reduced HiPIP I. The loop consists of residues 43-45 and the correlation time of the exchange process was determined to be 50 +/- 8 microseconds for the backbone nitrogen of Gln 44.
Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Espectroscopia de Ressonância Magnética/métodos , Complexo de Proteínas do Centro de Reação Fotossintética , Bactérias/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Glutamina/química , Proteínas Ferro-Enxofre/genética , Isótopos de Nitrogênio , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The solution structure of oxidized rat microsomal cytochrome b5 has been obtained from 1H NMR spectra measured at 800 MHz. The available assignment has been extended to 78% of the total protons and 95% of the residues. From 1372 meaningful NOEs, a family of 40 structures has been obtained through the program DYANA; 235 pseudocontact shifts have been then added as further constraints, obtaining an essentially similar family of structures. This latter family has been further refined through restrained energy minimization. The final RMSD values with respect to the average structure are 0.58 +/- 0.10 A and 1.05 +/- 0.11 A for backbone and heavy atoms, respectively. The high quality of the structure allows meaningful comparisons with the solution structure of the reduced protein, with the X-ray and solution structures of the oxidized bovine isoenzyme, and with the solution structure of the apoprotein. Upon loss of one electron, the heme plane undergoes a change in its orientation, possibly due to the change of the total charge. Propionate 7 appears to have a conformation which is dependent on the oxidation state of the iron. Helices alpha2 and alpha4 also experience changes in their average positions in the two oxidation states. Finally, the backbone NHs experience different exchange properties in the two oxidation states. While those present in the beta sheets forming the basis of the heme pocket are nonexchanging in both oxidation states, the NHs in the helices forming the heme-binding pocket are exchanging with the bulk solvent in the oxidized form, indicating larger local mobility in this state. This observation could suggest that, to optimize the electron transfer process, the local mobility should be properly tuned.
Assuntos
Citocromos b5/química , Citocromos b5/metabolismo , Microssomos Hepáticos/enzimologia , Sequência de Aminoácidos , Animais , Apoproteínas/química , Bovinos , Grupo dos Citocromos b/química , Citocromos b , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estrutura Secundária de Proteína , Prótons , Ratos , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
The mobility of (15)N labeled proteins can be characterized by measuring the cross-correlation rates delta(N,NI) that govern the conversion of Zeeman order N(z) of an amide (15)N nucleus into longitudinal two-spin order 2N(z)I(z) involving the amide (15)N and (1)H nuclei. This represents an alternative to the measurement of (15)N self-relaxation rates 1/T(1) and 1/T(2) or 1/T(1rho). The rate of interconversion between N(z) and 2N(z)I(z) is due to cross-correlation between fluctuations of different interactions and is not affected by a variety of relaxation mechanisms that contribute to the self-relaxation rates 1/T(1), 1/T(2) and 1/T(1rho). Spin diffusion among protons, which affects the measurements, can be quenched by various means that are evaluated by experiments and simulations. By applying an off-resonance radio-frequency (RF) field in the vicinity of the nitrogen resonance, the spectral density function J(omega) can be determined at the frequency origin and at the nitrogen Larmor frequency. The methods are applied to the paramagnetic High-Potential Iron-Sulfur Protein iso I (HiPIP I) from E. halophila in its reduced state.
RESUMO
The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4S]2+ polymetallic center has been fully labeled with 15N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eur. J. Biochem. 225, 715-725]. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and 15N-1H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180 degrees pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (rho para) has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and rho para data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the rho para constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH alpha and 37 3JHNC' (providing 45 phi dihedral angle constraints) 54 3JH alpha H beta and 31 3JNH beta (providing 26 chi 1 dihedral angle constraints), 4 chi 2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the beta CH protons, and 58 rho para constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.
Assuntos
Proteínas de Bactérias/química , Chromatiaceae/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Isótopos de Carbono , Chromatiaceae/genética , Hidrogênio/química , Proteínas Ferro-Enxofre/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Isótopos de Nitrogênio , Oxirredução , Conformação Proteica , Soluções , TermodinâmicaRESUMO
We have accounted for the effect of paramagnetism on the intensities of NOEs in a 73-residue paramagnetic metalloprotein, the reduced high-potential iron sulfur protein ISO I from Ectothiorhodospira halophila, whose solution structure had been recently solved by us. The paramagnetic effects were dealt with through a suitably modified complete relaxation matrix approach. We have then recalculated the structure through a distance geometry program by minimizing the difference between the sixth roots of the calculated and experimental NOEs. The average RMSD, calculated on residues 4-71, within the structures constituting the two families decreased from 0.67 to 0.46 angstrom for backbone atoms and from 1.23 to 1.06 angstroms for all heavy atoms. The structures in the new family are for the most part within the indetermination of the previous, less resolved, family. A few specific differences are detected and related to the presence of non-negligible paramagnetic effects, which are now properly evaluated.
Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Simulação por Computador , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oxirredução , Conformação Proteica , SoluçõesRESUMO
Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.
Assuntos
Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Tirosina/química , Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Sequência Conservada , Eletroquímica , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Proteínas Ferro-Enxofre/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , TermodinâmicaRESUMO
A 1H and 15N NMR investigation through two-dimensional and three-dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high-potential iron-sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are paramagnetic with a relatively low mu eff of about 0.8 mu B/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closet to the paramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X-ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date. Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter-residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X-ray crystallographic analysis of the wild-type protein at 250-pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild-type protein. These data are meaningful for the detection of structural differences among mutants in future studies.
Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Expressão Gênica , Proteínas Ferro-Enxofre/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Isótopos de Nitrogênio , Oxirredução , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TransfecçãoRESUMO
The three-dimensional structure in solution of reduced recombinant high-potential iron-sulfur protein iso-I from Ectothiorhodospira halophila was determined using 948 relevant interproton NOEs out of the 1246 observed NOEs. The determination was accomplished using the XEASY program for spectral analysis and the distance geometry (DG) program DIANA for generation of the structure as described by Wüthrich [Wüthrich, K. (1989) Acc. Chem. Res. 22, 36-44]. The FeS cluster was simulated using an amino acid residue constructed for the present work from a cysteinyl residue with an iron and a sulfur atom attached to the terminal thiol. The family of structures obtained from distance geometry were subjected to energy minimization and molecular dynamics simulations using previously defined force field parameters. The quality of these structures at each stage of the refinement process is discussed with respect to the dihedral angle order parameter and the root-mean-square deviation of the atomic coordinates. The latter values for the backbone atoms vary from 67 pm for the distance-geometry structures to 60 pm for the energy-minimized structures to 51 pm for the structures subjected to restrained molecular dynamics. Finally, the structure in best agreement with the NOE constraints has been further treated with extensive restrained molecular dynamics in water. The solution structure is well defined and is very similar to the available X-ray structure. We do not know of any previous determination of the structure of a paramagnetic protein in solution by NMR. The effect of paramagnetism on the quality of the structure determination is discussed.
Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Estrutura Terciária de Proteína , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Magnetismo , Estrutura Molecular , Conformação Proteica , SoluçõesRESUMO
The hyperfine couplings of the remote nitrogens of histidine ligands are determined for the first time by an X-band ESEEM spectroscopy study of 15N-substituted superoxide dismutase (SOD). They show a significant difference between two groups of ligands with different orientation relative to the metal ion. The ESEEM spectra of 15N SOD with cyanide as an inhibitor containing 14N and 15N are also discussed. They allow some conclusions to be drawn about structural changes upon inhibitor binding and indicate the necessity of further multifrequency investigations.