Biomedical subjects

C Luchinat

Publications and source records attributed to C Luchinat.

At least 37 records · Page 2Linked to original sources

PubMed2000

Protein hydration and location of water molecules in oxidized horse heart cytochrome c by (1)H NMR.

The hydration properties of the oxidized form of horse heart cytochrome c have been studied by (1)H NMR spectroscopy. Two-dimensional, homonuclear ePHOGSY-NOESY experiments are used to map water-protein interactions. The detected NOEs reveal interactions between nonexchangeable protein protons and both water protons and labile protein protons which exchange with water protons. Among the many water molecules apparent in the X-ray structure, three have been identified with a residence time longer than 300 ps. One of them is located inside the distal heme cavity, in the deepest part of a hydration pathway extending toward the surface. The identification of hydrophilic regions and detection of three long-lived water molecules settles some ambiguities and provides a better representation of the water-protein interactions in oxidized cytochrome c.

Animals↗

PubMed2000

Paramagnetic 1H NMR spectroscopy of the reduced, unbound photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed- and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA- and FB-.

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn. can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

Cyanobacteria↗

PubMed2000

15N chemical shift changes in cytochrome b5: redox-dependent vs. guanidinium chloride-induced changes.

The origin of the recently reported chemical shift changes of backbone amide nitrogens of redox proteins upon redox state changes has been investigated. These effects are particularly marked in cytochromes and are clearly present after correction for pseudocontact shifts in the oxidized form (Boyd J, Dobson CM, Morar AS, Williams RJP, Pielak GJ (1999) J Am Chem Soc 121:9247-9248; Guiles RD, Basus VJ, Sarma S, Malpure S, Fox KM, Kuntz ID, Waskell L (1993) Biochemistry 32:8329-8340). 15N-HSQC experiments have been performed on both oxidized and reduced forms of cytochrome b5 in the absence and in the presence of 2 M guanidinium chloride (GdmCl). GdmCl in this concentration is known to sizably alter the structure of the oxidized form of the protein and, in particular, to perturb the hydrogen bonding network. However, the perturbation of the 15N-NMR chemical shift changes is minor compared to the changes occurring upon reduction. It is concluded that changes in hydrogen bonding upon reduction must be modest and cannot account for the observed chemical shift effects. Alternative explanations should thus be looked for.

Animals↗

PubMed2000

Lanthanide induced residual dipolar couplings for the conformational investigation of peripheral 15NH2 moieties.

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.

Anisotropy↗

PubMed2000

High resolution solution structure of the protein part of Cu7 metallothionein.

The three-dimensional solution structure of the protein part of Cu7 metallothionein (Cu7MT) of Saccharomyces cerevisiae has been attempted by 1H two-dimensional NMR spectroscopy at 800 MHz. The protein part constitutes 53 amino acids. A total of 1192 NOEs, of which 1048 are meaningful, were used to determine the solution structure of the first 40 residues, the last 13 residues being disordered. A family of 30 structures was generated. Root-mean-square deviation (rmsd) values from the average structure of 0.32 +/- 0.13 A and 0.61 +/- 0.15 A for backbone and all heavy atoms, respectively, were obtained for the residues 2-40. The ten copper-coordinating cysteine sulfurs and the empty spaces around them are well defined. The structure of the protein part is similar but not identical to the available ones of the same holoprotein and of the Ag7 metallothionein, and is qualitatively superior. If the same metal-sulfur connectivities reported in the literature from 1H-109Ag heteronuclear multiple quantum coherence spectroscopy are assumed to hold for the present copper derivative, a peptide structure is obtained which is again similar, but still not identical, within indetermination, to that available. The structure of the copper polymetallic center may well be different from that proposed for the silver derivative, and indeed a number of different arrangements of the seven copper ions are consistent with the present highly refined structure of the protein part.

Amino Acid Sequence↗

PubMed1999

NMR characterization of substrate binding in the phthalate dioxygenase system.

The paramagnetic enhancements in the NMR relaxation rates for the fluorine in fluorophthalates have been used to determine the position of the phthalate with respect to the mononuclear metal ion in native and metal-substituted derivatives of phthalate dioxygenase (PDO). These studies show directly that the substrate interacts with the mononuclear metal of PDO and provide the first structural characterization of this interaction. With a molecular mass of 200 kDa, PDO is one of the largest proteins studied to date by paramagnetic NMR. Two paramagnetically broadened (19)F lines were observed for monofluorophthalates bound to CoPDO. This demonstrates that fluorophthalate binds to PDO with a handedness, i.e., with the fluorine label facing to the "right" or to the "left", relative to the hyperfine tensor of the Co(II). The relative affinities of the two orientations are slightly different, with a 2-fold and 5-fold excess of the preferred orientation for 4-fluorophthalate and 3-fluorophthalate, respectively. The longitudinal relaxation rate (T(1)) and transverse relaxation rate (T(2)) data give mutually consistent fluorine to cobalt distances. These results are consistent with approximate bilateral symmetry, with the Co to 3-fluorophthalate distances ( approximately 5.5 A) approximately 25% longer than the Co to 4-fluorophthalate distances ( approximately 4. 5 A). A detailed geometric model is derived from these data. This structural characterization of the mononuclear site provides a framework to develop hypotheses for the mechanism of oxygenation by the Fe(II)-containing aromatic dioxygenases.

Binding Sites↗

PubMed1999

Structural and dynamical properties of a partially unfolded Fe4S4 protein: role of the cofactor in protein folding.

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of 15N and 1H resonances, NOE data, proton longitudinal relaxation times, and 3JHNHalpha coupling constants as well as 15N relaxation parameters (T1, T2, T1rho, and 1H-15N NOE) were obtained and used to build a structural model of the intermediate. The Fe4S4 cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.

Iron-Sulfur Proteins↗

PubMed1999

Non-ionic bulky Gd(III) DTPA-bisamide complexes as potential contrast agents for magnetic resonance imaging.

A series of new diethylene triamine pentaacetic acid (DTPA)-bisamide chelates containing bulky alkyl and aryl side chains have been prepared and characterized. Nuclear magnetic relaxation dispersion profiles were measured for the neutral gadolinium [Gd(III)] DTPA-bisamide complexes in water solution, and their chemical exchange times (tau(m)) were found to be in the range of 1.4 to 4.9 micros. Significant enhancements of solvent proton relaxation rates were observed between 10 and 50 MHz for one of the complexes of the series [Gd(III)-DTPA-bis-2-ethylhexylamide] in human serum albumin (HSA) solution, indicating the formation of a paramagnetic macromolecular adduct. The binding association constant K(A) of the complex and the albumin 5.7 x 10(3) M(-1) was obtained, and the relaxivity of the fully bound adduct was determined to be 13.8 mM(-1) s(-1) at 20 MHz and 25 degrees C. The high value of K(A) makes the above derivative a good potential blood pool contrast agent at the physiological HSA concentration.

Contrast Media↗

PubMed1999

A low frequency 1H-NMR external unit for the analysis of large foodstuff samples.

An inexpensive external unit that allows the use of a commercial high-resolution NMR spectrometer as a very low frequency instrument is described. The external unit is phase coherent, the pulse timing being given by the parent spectrometer. With the exception of the probe, the external unit does not contain any tuned elements. This permits easy change of frequency in the range 100 kHz-1 MHz. The external unit may be appropriately employed in food science where, in several cases, low frequency is desirable. An application to hen shell eggs at the frequency of 700 kHz is described.

Animals↗

PubMed1999

Heme methyl 1H chemical shifts as structural parameters in some low-spin ferriheme proteins.

The different paramagnetic shifts of the four methyl groups in ferriheme proteins have been described as being due to the effect of the axial ligand nodal plane orientation. An equation, heuristically found and theoretically explained, describing the relation between contact and pseudocontact shifts and the position of the axial ligand(s) has been derived for bis-histidine ferriheme proteins and for cyanide-histidine ferriheme proteins. The values of the heuristic parameters contained in the equations were found by fitting the shifts of bovine cytochrome b5 and several bis-histidine cytochromes c3 and histidine-cyanide systems. The agreement between the observed and the calculated shifts was found to be good. Therefore, by taking advantage of this study, information on the position of the axial ligands, that can be used as a constraint for structure determination, can be obtained from the shifts of the methyl protons.

Animals↗

PubMed1999

Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP.

The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, one of the cluster ligands. The reduction potential of the S79P variant was 104 +/- 3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins. These results suggest that the influence of amide group 79 on the reduction potential of C. vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.

Base Sequence↗

PubMed1999

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I from Ectothiorhodospira halophila.

Mössbauer spectra of the oxidized [Fe4S4]3+ and the reduced [Fe4S4]2+ clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and 57Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe4S4]3+ clusters, the Mössbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe3+ atoms couple to the "ferric-ferric" pair, and one Fe2+ and one Fe3+ atom give the "ferric-ferrous pair". For the simulation of the Mössbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by 57Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of Mössbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical Mössbauer spectra. Spin-spin and spinlattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the Mössbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the Mössbauer spectrum, indicating a mixed-valence state of Fe2.5+ for each.

Bacterial Proteins↗

PubMed1999

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli.

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S](+) form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S](+) form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S](2+), [3Fe-4S](+/0), and to the stable [2Fe-2S](2+) form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Furthermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.

Anaerobiosis↗

PubMed1999

New applications of paramagnetic NMR in chemical biology.

The methodological accessibility to solution structure and dynamic investigation of paramagnetic metallobiomolecules has afforded the ability to tackle the redox pairs of electron transfer proteins of which at least one is paramagnetic, to study the orientation effects of high magnetic fields on paramagnetic biomolecules, and finally to study the role of metal-based cofactors in protein folding and stability.

Magnetic Resonance Spectroscopy↗

PubMed1999

Ab initio solution and refinement of two high-potential iron protein structures at atomic resolution.

The crystal structure of the reduced high-potential iron protein (HiPIP) from Chromatium vinosum has been redetermined in a new orthorhombic crystal modification, and the structure of its H42Q mutant has been determined in orthorhombic (H42Q-1) and cubic (H42Q-2) modifications. The first two were solved by ab initio direct methods using data collected to atomic resolution (1.20 and 0. 93 A, respectively). The recombinant wild type (rc-WT) with two HiPIP molecules in the asymmetric unit has 1264 protein atoms and 335 solvent sites, and is the second largest structure reported so far that has been solved by pure direct methods. The solutions were obtained in a fully automated way and included more than 80% of the protein atoms. Restrained anisotropic refinement for rc-WT and H42Q-1 converged to R(1) = summation operator||F(o)| - |F(c)|| / summation operator|F(o)| of 12.0 and 13.6%, respectively [data with I > 2sigma(I)], and 12.8 and 15.5% (all data). H42Q-2 contains two molecules in the asymmetric unit and diffracted only to 2.6 A. In both molecules of rc-WT and in the single unique molecule of H42Q-1 the [Fe(4)S(4)](2+) cluster dimensions are very similar and show a characteristic tetragonal distortion with four short Fe-S bonds along four approximately parallel cube edges, and eight long Fe-S bonds. The unique protein molecules in H42Q-2 and rc-WT are also very similar in other respects, except for the hydrogen bonding around the mutated residue that is at the surface of the protein, supporting the hypothesis that the difference in redox potentials at lower pH values is caused primarily by differences in the charge distribution near the surface of the protein rather than by structural differences in the cluster region.

Animals↗

PubMed1998

The solution structure of parsley [2Fe-2S]ferredoxin.

The [2Fe-2S]ferredoxin I (Fd I) from parsley leaves (Mr = 10,500; 96 amino acids) in the Fe(III)-Fe(III) oxidized form has been studied by 1H-NMR spectroscopy. Sequence-specific 1H-NMR assignments were obtained through two-dimensional classical double-quantum-filtered-COSY, NOESY and TOCSY spectra. NOEs between protons as close as 5.6 A from the paramagnetic Fe(III) atoms were observed at 800 MHz. A total of 3066 NOEs (of which 2533 are meaningful) and 18 distance constraints taken from X-ray crystallography of the Fe2S2 active site were used to obtain the solution structure. From inversion recovery NOESY experiments, 33 longitudinal relaxation rate (Qpara) constraints were used for the structural refinement. The final structure was obtained by a process of restrained energy minimization. Root-mean-square (rmsd) deviation values obtained for the family of 18 structures (with reference to the average structure) are 0.52 +/- 0.10 A and 0.91 +/- 0.12 A for backbone and all heavy atoms respectively. The structure consists of seven-strands of beta-sheets and four short alpha-helices. The quality of the present solution structure is among the best of those reported for [2Fe-2S]ferredoxins. The secondary structure and overall folding are compared with those of Anabaena variabilis Fd and the higher plant Equistum arvense (horse tail) protein determined through X-ray crystallography. The groups believed to be responsible for electron transfer have been analysed.

Amino Acid Sequence↗

PubMed1998

Solution structure of an artificial Fe8S8 ferredoxin: the D13C variant of Bacillus schlegelii Fe7S8 ferredoxin.

The solution structure of the D13C variant of the thermostable Fe7S8 ferredoxin from Bacillus schlegelii has been determined by 1H-NMR spectroscopy in its oxidized form. In a variable-temperature NMR study the D13C variant was as thermostable (up to 90 degrees C) as the wild-type protein (WT). Seventy-five out of 77 amino acid residues and 81% of all theoretically expected proton resonances in the D13C Fe8S8 protein have been assigned. Its structure was determined through torsion angle dynamics calculations with the program DYANA, using 935 meaningful NOEs (from a total of 1251), hydrogen bond constraints, and NMR-derived dihedral angle constraints for the cluster-ligating cysteines. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.055 nm for the backbone atoms and of 0.099 nm for all heavy atoms. The overall folding of the WT is maintained in the mutant, except for the immediate vicinity of the new cysteine, which becomes much more similar to native Fe8S8 proteins. The two residues at positions 11 and 12, which constitute an insertion with respect to all known Fe8S8 proteins, assume a conformation that does not prevent the preceding and following residues from folding like in native Fe8S8 proteins. Clear evidence for the existence of two conformations involving almost half of the amino acid residues was found. The two conformations are structurally indistinguishable. Temperature-dependent NMR experiments show that one of them is thermodynamically more stable than the other.

Amino Acid Sequence↗

PubMed1998

Solution structure of the oxidized Fe7S8 ferredoxin from the thermophilic bacterium Bacillus schlegelii by 1H NMR spectroscopy.

The solution structure of the paramagnetic seven-iron ferredoxin from Bacillus schlegelii in its oxidized form has been determined by 1H NMR. The protein, which contains 77 amino acids, is thermostable. Seventy-two residues and 79% of all theoretically expected proton resonances have been assigned. The structure has been determined through torsion angle dynamics calculations with the program DYANA, using 966 meaningful NOEs (from a total of 1305), hydrogen bond constraints, and NMR derived dihedral angle constraints for the cluster ligating cysteines, and by using crystallographic information to build up the two clusters. Afterwards, restrained energy minimization and restrained molecular dynamics were applied to each conformer of the family. The final family of 20 structures has RMSD values from the mean structure of 0.68 A for the backbone atoms and of 1.16 A for all heavy atoms. The contributions to the thermal stability of the B. schlegelii ferredoxin are discussed by comparing the present structure to that of the less stable Azotobacter vinelandii ferredoxin I which is the only other available structure of a bacterial seven-iron ferredoxin. It is proposed that the hydrophobic interactions and the hydrogen bond network linking the N-terminus and the C-terminus together and a high number of salt bridges contribute to the stability.

Amino Acid Sequence↗