Biomedical subjects

M Bruschi

Publications and source records attributed to M Bruschi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106-126.

Prion diseases are marked by the cerebral accumulation of conformationally modified forms of the cellular prion protein (PrP(C)), known as PrP(res). The region comprising the residues 106-126 of human PrP seems to have a key role in this conformational conversion, because a synthetic peptide homologous with this sequence (PrP106-126) adopts different secondary structures in different environments. To investigate the molecular determinants of the physicochemical characteristics of PrP106-126, we synthesized a series of analogues including PrP106-126 H(D), PrP106-126 A and PrP106-126 K, with l-His-->d-His, His-->Ala and His-->Lys substitutions respectively at position 111, PrP106-126 NH(2) with amidation of the C-terminus, PrP106-126 V with an Ala-->Val substition at position 117, and PrP106-126 VNH(2) with an Ala-->Val substitution at position 117 and amidation of the C-terminus. The analysis of the secondary structure and aggregation properties of PrP106-126 and its analogues showed the following. (1) His(111) is central to the conformational changes of PrP peptides. (2) Amidation of the C-terminal Gly(126) yields a predominantly random coil structure, abolishes the molecular polymorphism and decreases the propensity of PrP106-126 to generate amyloid fibrils. (3) PrP106-126 V, carrying an Ala-->Val substitution at position 117, does not demonstrate a fibrillogenic ability superior to that of PrP106-126. However, the presence of Val at position 117 increases the aggregation properties of the amidated peptide. (4) Amyloid fibrils are not required for neurotoxicity because the effects of PrP106-126 NH(2) on primary neuronal cultures were similar to those of the wild-type sequence. Conversely, astroglial proliferation is related to the presence of amyloid fibrils, suggesting that astrogliosis in prion encephalopathies without amyloid deposits is a mediated effect rather than a direct effect of disease-specific PrP isoforms.

Amides↗

PubMed1999

Key role of phenylalanine 20 in cytochrome c3: structure, stability, and function studies.

Aromatic residues in c-type cytochromes might have an important function in the folding and/or electron transferring properties of the molecule. In the tetraheme cytochrome c3 (Mr 13 000) from Desulfovibrio vulgaris Hildenborough, Phe20, is located between heme 1 and heme 3 with its aromatic ring close and almost parallel to the ring plane of heme 1. We replaced this residue by a nonaromatic hydrophobe residue, leucine, and analyzed the effects in terms of functional, structural, and physicochemical properties. While the F20L replacement did not have any strong effects on the heme region stability, a decrease of the thermostability of the whole molecule was observed. In the same way, the four macroscopic redox potentials were affected by the mutation as well as the flexibility of the surface loop around heme 4. The F20L replacement itself and/or this structural modification might be responsible for the loss of the intermolecular cooperativity between F20L cytochrome c3 molecules.

Amino Acid Substitution↗

PubMed1999

Analytical titration curves of glycosyl hydrolase Cel45 by combined isoelectric focusing-electrophoresis.

The electrical charge of endocellulase Cel45-core has been determined by combined isoelectric focusing-electrophoresis in the range of pH 3-9. In order to transform electrophoretic mobility to absolute electrical charge value, several corrections were applied: the frictional coefficient theoretically calculated from the molecular dimensions depends on porous gel structure and on the ionic strength of the solution. By comparing the curve calculated according to the Linderstrom-Lang equation, the number of charged electrical groups exposed to the solvent and their apparent ionization constants, pK(o)i, can be determined. Furthermore, the macromolecule structure can be assumed not to change in this pH range. This finding is necessary to understand the structure and the electrical properties of the entire Cel45 molecule.

Aspergillus↗

PubMed1999

A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.

Amino Acid Sequence↗

PubMed1999

First evidence for the presence of a hydrogenase in the sulfur-reducing bacterium Desulfuromonas acetoxidans.

Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from Desulfuromonas acetoxidans. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium D. acetoxidans, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of D. acetoxidans, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of the gene coding for human cytidine deaminase.

The human gene coding for cytidine deaminase (CD), the enzyme which catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, was isolated and structurally characterized. CD is a single copy gene with a length of 31 kb and consists of four exons. Exon-intron junctions do not bracket functional domains of the encoded protein as the boundary between exons 2 and 3 interrupts the catalytically important zinc-finger domain, which is well conserved along phylogenesis. 5'-RACE and RNase mapping experiments identify one major and multiple other minor transcription initiation sites, which are present in placenta as well as in the myeloid cell lines, HL-60 and U937. The 5'-flanking region of the gene contains an orientation-dependent functional promoter and is characterized by the presence of several potential sites for the binding of known transcriptional factors.

Amino Acid Sequence↗

PubMed1998

Kinetics and interaction studies between cytochrome c3 and Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough.

Hydrogenases from Desulfovibrio are found to catalyze hydrogen uptake with low potential multiheme cytochromes, such as cytochrome c3, acting as acceptors. The production of Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough was improved with respect to the growth phase and media to determine the best large-scale bacteria growth conditions. The interaction and electron transfer from Fe-only hydrogenase to multiheme cytochrome has been studied in detail by both BLAcore and steady-state measurements. The electron transfer between [Fe] hydrogenase and cytochrome c3 appears to be a cooperative phenomenon (h = 1.37). This behavior could be related to the conductivity properties of multihemic cytochromes. An apparent dissociation constant was determined (2 x 10(-7) M). The importance of the cooperativity for contrasting models proposed to describe the functional role of the hydrogenase/cytochrome c3 complex is discussed. Presently, the only determined structure is from [NiFe] hydrogenase and there are no obvious similarities between [NiFe] and [Fe] hydrogenase. Furthermore, no crystallographic data are available concerning [Fe] hydrogenase. The first results on crystallization and X-ray crystallography are reported.

Biosensing Techniques↗

PubMed1998

Characterization and expression of the co-transcribed cyc1 and cyc2 genes encoding the cytochrome c4 (c552) and a high-molecular-mass cytochrome c from Thiobacillus ferrooxidans ATCC 33020.

The sequence of the cyc1 gene encoding the Thiobacillus ferrooxidans ATCC 33020 c552 cytochrome, shows that this cytochrome is a 21-kDa periplasmic c4-type cytochrome containing two similar monohaem domains. The kinetics of reduction and the fact that cytochromes c4 are considered to be physiological electron donors of cytochrome oxidases suggest that the last steps of the iron respiratory chain are: rusticyanin-->cytochrome c4-->cytochrome oxidase. In Thiobacillus ferrooxidans, cyc1 is co-transcribed with the cyc2 gene, encoding a high-molecular-mass monohaem cytochrome c. This suggests that the cytochromes encoded by these genes belong to the same electron transfer chain.

Amino Acid Sequence↗

PubMed1998

800 MHz 1H NMR solution structure refinement of oxidized cytochrome c7 from Desulfuromonas acetoxidans.

The solution structure of Desulfuromonas acetoxidans cytochrome c7 has been refined by using 1H-NMR spectra recorded at 800 MHz and by using pseudocontact shifts in the final energy minimization procedure. The protein, composed of 68 amino acids, contains three paramagnetic heme moieties, each with one unpaired electron. The largely distributed paramagnetism broadens the lines in several protein parts. The structure is now relatively well resolved all over the backbone by the use of 1315 meaningful NOEs and 90 pseudocontact shifts. The statistical analysis of the structure indicates its satisfactory quality. The protein-fold is quite similar to that of the analogous four-heme cytochromes c3 for those parts which can be considered homologous. The solvent accessibility and the electrostatic potential surfaces surrounding the three hemes have been analyzed in terms of their reduction potentials. The resulting magnetic susceptibility anisotropy data obtained from pseudocontact shifts are analyzed in terms of structural data.

Amino Acid Sequence↗

PubMed1998

Structural and kinetic studies of the Y73E mutant of octaheme cytochrome c3 (Mr = 26 000) from Desulfovibrio desulfuricans Norway.

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.

Binding Sites↗

PubMed1998

Characterization of the cytochromes C from Desulfovibrio desulfuricans G201.

A monoheme cytochrome c553 and a hexadecaheme high molecular weight cytochrome (Hmc) have been isolated and characterized from the sulfate-reducing bacteria Desulfovibrio desulfuricans G201, in addition to the tetraheme cytochrome c3 (Mr 13000) that has been previously described. Both cytochromes are homologous with respect to several biochemical properties to the corresponding cytochromes found in other Desulfovibrio species. However, they are acidic proteins while the corresponding molecules, isolated from other Desulfovibrio species, are relatively more basic. The D. desulfuricans cytochrome content appears identical to that of D. vulgaris Hildenborough. Isolation of these cytochromes from a Desulfovibrio desulfuricans strain is of great interest in order to get more insight on the physiological function of these molecules.

Amino Acid Sequence↗

PubMed1998

The Desulfuromonas acetoxidans triheme cytochrome c7 produced in Desulfovibrio desulfuricans retains its metal reductase activity.

Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7.

Amino Acid Sequence↗

PubMed1997

Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans.

The multimeric cytochromes described to date in sulfate- and sulfur-reducing bacteria are associated with diverse respiratory modes involving the use of elemental sulfur or oxidized sulfur compounds as terminal acceptors. They exhibit no structural similarity with the other cytochrome c classes and are characterized by a bis-histidinyl axial iron coordination and low redox potentials. We have purified two new cytochromes c with markedly different molecular masses (10 000 and 50 000) from the bacterium Desulfuromonas acetoxidans, which uses anaerobic sulfur respiration as its sole energy source. The characterization by electrochemistry and optical and EPR spectroscopies revealed the cytochrome c (Mr = 10 000) to be the first monohemic cytochrome c exhibiting a bis-histidinyl axial coordination and a low redox potential (-220 mV). The cytochrome c (Mr = 50 000) contains four hemes of low potential (-200, -210, -370, and -380 mV) with the same axial coordination. The N-terminal amino acid sequences were compared with that of the trihemic cytochrome c7, previously described in D. acetoxidans and which is related to tetrahemic cytochrome c3 from sulfate reducing bacteria. Some homology was found between cytochrome c (Mr = 10 000) and cytochrome c7. Both D. acetoxidans cytochromes c are located in the periplasmic space and their biochemical and spectroscopic properties indicate that they belong to the class III cytochromes.

Amino Acids↗

PubMed1997

A single mutation in the heme 4 environment of Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000) greatly affects the molecule reactivity.

The gene encoding Desulfovibrio desulfuricans Norway cytochrome c3 (Mr 26,000), a dimeric octaheme cytochrome belonging to the polyheme cytochrome c3 superfamily, has been cloned and successfully expressed in another sulfate reducing bacteria, D. desulfuricans G201. The gene, named cycD, is monocistronic and encodes a cytochrome precursor of 135 amino acids with an extension at the NH2 terminus of 24 amino acids. This extension acts as a signal sequence which allows export across the cytoplasmic membrane into the periplasmic space. Tyrosine 73, which is in a close contact with the histidine sixth axial ligand to the heme 4 iron atom, has been replaced by a glutamate residue using site-directed mutagenesis. The cytochrome mutant when expressed in D. desulfuricans G201, is correctly folded and matured. A global increase of the oxidoreduction potentials of about 50 mV is measured for the Y73E cytochrome. The mutation also has a strong influence on the interaction of the cytochrome with its redox partner, the hydrogenase. This suggests, like the tetraheme cytochrome c3 (Mr 13, 000), heme 4 is the interactive heme in the cytochrome-hydrogenase complex and that alteration of the heme 4 environment can greatly affect the electron transfer reaction with its redox partner.

Amino Acid Sequence↗

PubMed1996

The cytochrome c3 superfamily: amino acid sequence of a dimeric octahaem cytochrome c3 (M(r) 26,000) isolated from Desulfovibrio gigas.

Cytochrome c3 (M(r) 26000) isolated from Desulfovibrio gigas is a dimeric cytochrome consisting of two identical subunits of 109 amino acids, each of which contains four haem groups. On the basis of its amino acid sequence, this cytochrome clearly belongs to the cytochrome c3 superfamily, and will be classified in class III of the c-type cytochromes as defined by Ambler [(1980) in From Cyclotrons to Cytochromes (Robinson, A. B. and Kaplan, N. O., eds.), pp. 263-279, Academic Press, London]. It contains ten cysteine and nine histidine residues in each subunit, and eight cysteines and eight histidines linked to the four haem groups were found to be invariant on alignment of all known cytochrome c3 sequences. Two intermolecular disulphide bridges have been determined between cysteine residues 5 and 46 of the two monomers. Cytochrome c3 (M(r) 26,000) from D gigas is clearly different from cytochrome c3 (M(r) 13,000) from the same strain, with which it shows only 27% sequence identity. Compared with cytochrome c3 (M(r) 26,000) from D. desulfuricans Norway, the three-dimensional structure of which has been determined, 26.95% of the residues have been conserved. In the enzyme from D. desulfuricans Norway, hydrophobic interactions have been described across the dimer interface. Residues involved in similar interactions seem to be well conserved in the equivalent D. gigas cytochrome. This sequence provides structural data to allow specification of this new subclass of polyhaem cytochromes. Furthermore, D. gigas cytochrome c3 (M(r) 26,000) is the first polyhaem cytochrome shown to contain two disulphide bridges linking two identical subunits, which could induce more rigid folding. The folding and the evolution of this family of polyhaem cytochromes are discussed.

Amino Acid Sequence↗

PubMed1996

NMR characterization and solution structure determination of the oxidized cytochrome c7 from Desulfuromonas acetoxidans.

The solution structure of the three-heme electron transfer protein cytochrome c7 from Desulfuromonas acetoxidans is reported. The determination of the structure is obtained through NMR spectroscopy on the fully oxidized, paramagnetic form. The richness of structural motifs and the presence of three prosthetic groups in a protein of 68 residues is discussed in comparison with the four-heme cytochromes c3 already characterized through x-ray crystallography. In particular, the orientation of the three hemes present in cytochrome c7 is similar to that of three out of four hemes of cytochromes c3. The reduction potentials of the individual hemes, which have been obtained through the sequence-specific assignment of the heme resonances, are discussed with respect to the properties of the protein matrix. This information is relevant for any attempt to understand the electron transfer pathway.

Amino Acid Sequence↗

PubMed1996

Characterisation of a soluble cytochrome c4 isolated from Thiobacillus ferrooxidans.

A soluble c-type cytochrome was purified to homogeneity from Thiobacillus ferrooxidans. This cytochrome is characterised by an alpha-peak wavelength of 552 nm, a molecular mass of 21 193 Da (as determined by mass spectroscopy), and a pI value of 9. N-terminal sequencing yielded the polypeptide sequence up to the 50th residue. The iron content of 1.9 Fe/molecule and the heme/molecule ratio of 2.15 identified this cytochrome as a diheme protein. Optical redox titrations at pH 3.0 revealed the presence of two distinguishable redox species with Em = 385 mV +/- 20 mV and Em = 480 mV +/- 20 mV. EPR spectra recorded on this heme protein showed the presence of two distinct spectral species with gz = 3.1 and gz = 3.35. The gz = 3.35 heme corresponds to the higher potential redox species. In line with the differences in Em values, the two heme species were oxidised by O2 with significantly differing half-times. All the above mentioned properties demonstrate that this heme protein belongs to the c4 family of diheme cytochromes. The characteristics and functional role of the studied heme protein are discussed with reference to other c-type cytochromes described in Thiobacilli. Its properties are furthermore compared to other members of the cytochrome c4 family.

Amino Acid Sequence↗

PubMed1996

Electron transfer in tetrahemic cytochromes c3: spectroelectrochemical evidence for a conformational change triggered by heme IV reduction.

Electron transfer in tetrahemic cytochromes c3 from Desulfovibrio vulgaris Hildenborough (D.v.H.) and Desulfovibrio desulfuricans Norway (D.d.N.) strains has been investigated by thin layer spectroelectrochemistry with visible absorption, CD, and resonance Raman (RR) monitoring. The observed splitting of the isosbestic point in the Soret absorption band indicates that the electron transfer from the (FeIII)4 state to the (FeII)4 state proceeds via an intermediate species, which corresponds to 25 and 50% reduction for the D.v.H. cyt.c3 and the D.d.N. cyt.c3, respectively. For the latter, a specific CD signal is observed at half-reduction. RR monitoring of the redox process does not reveal multiple splitting of the high-frequency RR bands, at variance with previously published results on the enzymatic reduction of cyt.c3 from Desulfovibrio vulgaris Miyazaki, a cytochrome highly homologous to D.v.H. cyt.c3 [Verma, A.L., Kimura, A., Nakamura, A., Yagi, T., Inoguchi, H., & Kitagawa, T. (1988) J. Am. Chem. Soc. 110, 6617-6623]. The low-frequency RR spectra of the intermediate species differ significantly from the ones calculated from a linear combination of the all-ferric and all-ferrous states, for the same reduction ratio. Frequency shifts of the bending modes of the cysteine and propionate heme substituents are observed, as well as changes specific to each cytochrome; most notable is the activation of two torsional modes in the case of D.d.N. cyt.c3. Comparison of the results obtained for the two cytochromes leads to the conclusion that reduction of heme IV triggers the observed conformational change. This conclusion is supported by the spectroelectrochemical investigation of the mutant D.v.H. cyt.c3 H25M, in which the sixth ligand of heme III, histidine, is replaced by a methionine.

Circular Dichroism↗