PubMed Health⌕ Search

Biomedical subjects

S Samar Hasnain

Publications and source records attributed to S Samar Hasnain.

30 records · Page 2Linked to original sources
PubMed2003

The structure of glyceraldehyde 3-phosphate dehydrogenase from Alcaligenes xylosoxidans at 1.7 A resolution.

The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the Gram-negative denitrifying bacterial species Alcaligenes xylosoxidans was purified and crystallized as a contaminant protein during purification of nitrous oxide reductase. This is the first structure of a GAPDH from a denitrifying species. The crystal structure was solved at 1.7 A resolution by molecular replacement using the structure of GAPDH from Bacillus stearothermophilus as a starting model. The quality of the structure enabled the amino-acid sequence of the A. xylosoxidans GAPDH to be assigned. The structure is that of the apo-enzyme, lacking the NAD+ cofactor and with the active-site residue Cys154 oxidized. The global structure of the enzyme has a homotetrameric quaternary structure similar to that observed for its bacterial and eukaryotic counterparts. The essential role of Cys154 in the enzyme activity has been confirmed. In monomer O two half-occupancy sulfate ions were found at the active site, which are analogous to the substrate and the "attacking" phosphate seen in B. stearothermophilus. One half-occupancy sulfate ion is also located in the substrate-binding site of monomer P.

Achromobacter denitrificans↗
PubMed2003

Structure of fully reduced bovine copper zinc superoxide dismutase at 1.15 A.

Copper zinc superoxide dismutase (CuZnSOD) forms a crucial component of the cellular response to oxidative stress by catalyzing the dismutation of the superoxide radical to hydrogen peroxide and water. Mutations in human CuZnSOD are associated with the development of familial amyotrophic lateral sclerosis (motor neuron disease). We have determined the structure of fully reduced bovine CuZnSOD to 1.15 A, the only atomic resolution structure for an intact CuZnSOD and one of only a small number for metalloproteins. For the first time, both subunits have been captured with the three coordinate Cu(I) ligation required by the generally accepted catalytic mechanism, where dismutation of the superoxide radical occurs via reduction of Cu. Furthermore, the improved resolution compared to previous studies (to 1.65 A) has allowed a more detailed examination of the metal center environment and its associated water network in the active site channel, facilitating the analysis of potential proton transfer routes.

Animals↗
PubMed2003

Amyloid-like filaments and water-filled nanotubes formed by SOD1 mutant proteins linked to familial ALS.

Mutations in the SOD1 gene cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human FALS patients and in transgenic mice expressing these mutant proteins, aggregates containing FALS SOD1 are observed. Accumulation of SOD1 aggregates is believed to interfere with axonal transport, protein degradation and anti-apoptotic functions of the neuronal cellular machinery. Here we show that metal-deficient, pathogenic SOD1 mutant proteins crystallize in three different crystal forms, all of which reveal higher-order assemblies of aligned beta-sheets. Amyloid-like filaments and water-filled nanotubes arise through extensive interactions between loop and beta-barrel elements of neighboring mutant SOD1 molecules. In all cases, non-native conformational changes permit a gain of interaction between dimers that leads to higher-order arrays. Normal beta-sheet-containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions. Loss of this protection through conformational rearrangement in the metal-deficient enzyme could be a toxic property common to mutants of SOD1 linked to FALS.

Amyloid↗
PubMed2002

XAFS studies of nitrogenase: the MoFe and VFe proteins and the use of crystallographic coordinates in three-dimensional EXAFS data analysis.

This paper reports a three-dimensional EXAFS refinement of the Mo coordination sphere of the FeMoco cluster of the dithionite-reduced MoFe protein from Klebsiella pneumoniae nitrogenase (Kp1) using the 1.6 A-resolution crystallographic coordinates. At this resolution, the positions of the heavy (Fe and S) atoms of the cluster are well determined and there is excellent agreement between the crystallographic and EXAFS models. However, the lighter homocitrate and histidine ligands are poorly determined in the crystal structure, and it is shown that the application of EXAFS-derived distance restraints during the early stages of crystallographic refinement provides a means of substantially improving (by approximately 0.1 A) the final crystallographic model. The consistency of the EXAFS analysis with the crystallographic information in this case justifies applications of EXAFS to cases where protein crystal structures are absent. Thus, the VFe protein of V-nitrogenase has been shown by EXAFS to possess a V-atom site catalytically similar to the well characterized MoFe-nitrogenases, with V replacing Mo.

Absorptiometry, Photon↗
PubMed2002

Introductory overview: X-ray absorption spectroscopy and structural genomics.

A special Issue of the Journal is presented, dedicated to biological applications of X-ray absorption spectroscopy (BioXAS) and examining the role of this technique in post-genomic biology. The Issue confirms that BioXAS has come of age and it can be expected to make a significant contribution in the structural genomics effort on metalloproteins, which are estimated to make up about 30% of proteins coded by genomes.

Absorptiometry, Photon↗
PubMed2002

Marriage of XAFS and crystallography for structure-function studies of metalloproteins.

Metalloproteins constitute a significant proportion of all known genomes. In this Article it is shown that the marriage of two synchrotron-radiation-based structural techniques, namely high-resolution crystallography and XAFS, can yield unprecedented levels of structural information that is directly relevant to the mechanism/function of these important macromolecules.

Absorptiometry, Photon↗
PubMed2002

Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin.

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.

Alcaligenes↗
PubMed2002

Crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin from Thiobacillus ferrooxidans: insights into the structural relationship with the cupredoxins and the multi copper proteins.

The crystal structures of the Met148Leu and Ser86Asp mutants of rusticyanin are presented at 1.82 and 1.65 A resolution, respectively. Both of these structures have two molecules in the asymmetric unit compared to the one present in the crystal form of the native protein. This provides an opportunity to investigate intramolecular electron transfer pathways in rusticyanin. The redox potential of the Met148Leu mutant ( approximately 800 mV) is elevated compared to that of the native protein ( approximately 670 mV at pH 3.2) while that of the Ser86Asp mutant ( approximately 623 mV at pH 3.2) is decreased. The effect of the Ser86Asp mutation on the hydrogen bonding near the type 1 Cu site is discussed and hence its role in determining acid stability is examined. The type 1 Cu site of Met148Leu mimics the structural and biochemical characteristics of those found in domain II of ceruloplasmin and fungal laccase. Moreover, the native rusticyanin's cupredoxin core and the type 1 Cu site closely resemble those found in ascorbate oxidase and nitrite reductase. Structure based phylogenetic trees have been re-examined in view of the additional structural data on rusticyanin and fungal laccase. We confirm that rusticyanin is in the same class as nitrite reductase domain 2, laccase domain 3 and ceruloplasmin domains 2, 4 and 6.

Amino Acid Sequence↗
PubMed2002

The N-terminal extension of rusticyanin is not responsible for its acid stability.

The N-terminal extension of rusticyanin is a unique structural feature of this protein in the cupredoxin family and has been speculated to be responsible for the extreme acid stability of the protein. We have removed the 35 residues from the N-terminus and show that the resulting -35 mutant is insoluble in aqueous media above pH 5.0 and exists primarily in a hexameric form at lower pHs. Synchrotron radiation circular dichroism (SRCD) and solution X-ray scattering data indicate that much of the beta-sheet structure is retained in acidic solution and indeed there is a small but significant increase in the beta-sheet contribution. We suggest this to be a result of beta-sheet formation between the monomer interfaces. The mutant does not bind copper. These results provide evidence that the unique N-terminus of rusticyanin is not responsible for the acid stability of the hydrophobic beta-barrel core of the protein.

Amino Acid Sequence↗
PubMed2002

Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism.

Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO(2)(-)) to nitric oxide (NO). Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 A, 1.9 A and 2.2 A resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme.

Alcaligenes↗
PubMed2002

Electron donation between copper containing nitrite reductases and cupredoxins: the nature of protein-protein interaction in complex formation.

In denitrifying organisms with copper containing dissimilatory nitrite reductases, electron donation from a reduced cupredoxin is an essential step in the reduction of nitrite to nitric oxide. Copper nitrite reductases are categorised into two subgroups based on their colour, green and blue, which are found in organisms where the cupredoxins are pseudoazurins and azurins, respectively. In view of this and some in vitro electron donation experiments, it has been suggested that copper nitrite reductases have specific electron donors and that electron transfer takes place in a specific complex of the two proteins. We report results from the first comprehensive electron donation experiments using three copper nitrite reductases, one green and two blue, and five cupredoxins, one pseudoazurin and four azurins. Our data show that pseudoazurin can readily donate electrons to both blue and green copper nitrite reductases. In contrast, all of the azurins react very sluggishly as electron donors to the green nitrite reductase. These results are discussed in terms of surface compatibility of the component proteins, complex formation, overall charges, charge distribution, hydrophobic patches and redox potentials. A docking model for the complexes is proposed.

Azurin↗