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S S Hasnain

Publications and source records attributed to S S Hasnain.

At least 55 records · Page 3Linked to original sources
PubMed1996

Direct-method structure determination of the native azurin II protein using one-wavelength anomalous scattering data.

The one-wavelength anomalous scattering (OAS) X-ray diffraction data of azurin II, a copper-containing protein from Alcaligenes xylosoxidans were collected at the Photon Factory, Japan at a 'routine' wavelength of 0.97 A. The structure had been originally solved by the molecular-replacement method [Dodd, Hasnain, Abraham, Eady & Smith (1995). Acta Cryst. D51, 1052-1064]. As a technique of ab initio structure determination, the direct method [Fan, Hao, Gu, Qian, Zheng & Ke (1990). Acta Cryst. A46, 935-939] was attempted to break the phase ambiguity intrinsic to OAS data. The phases were then improved using the solvent-flattening method. The final electron-density map clearly shows most Calpha positions and many side chains and it is traceable without prior knowledge of the structure. It is concluded that the direct method is capable of phasing anomalous scattering data collected at one wavelength from moderate-sized native proteins (M(w) approximately 20 kDa) which contain copper or atoms with a similar scattering power.

Journal Article↗
PubMed1996

State-of-the-Art Analysis of Whole X-ray Absorption Spectra.

Fitting an entire X-ray spectrum rather than its components, EXAFS and XANES, has been an aim of the practitioners of these techniques. Recent developments have made the calculations of both the scattering and atomic components practicable. We present the analysis of four representative model compounds using the EXCURVE package, which was modified to undertake this. The details of these modifications are also given. A comparison of matrix-inversion and finite-path-sum methods is made which shows that the latter method is more promising for fitting the edge region. A number of enhancements are required before this approach can be used for accurate structure determination. These include improvement in atomic contribution, better potentials/phase shifts, and the ability to calculate and refine multiple-scattering terms to at least fifth order.

Journal Article↗
PubMed1996

The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene.

The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced. A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers. The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence. The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli.

Amino Acid Sequence↗
PubMed1996

Structure Solution of Azurin II from Alcaligenes xylosoxidans using the Laue Method: Possibility of Studying In Situ Redox Changes using X-rays.

We have recently demonstrated that X-rays can be used for changing the redox states of the metal centre in metalloproteins [Murphy et al. (1995). J. Synchrotron Rad. 2, 64-69]. The possibility of using the Laue method for studying the structural changes associated with such X-ray-induced reactions is explored by applying the method to the structure determination of a new azurin (hereafter referred to as azurin II) from the denitrifying bacterium Alcaligenes xylosoxidans. Laue X-ray diffraction data of azurin II were collected at station 9.7 of the SRS Daresbury. Three diffraction patterns were recorded on film packs at three different crystal orientations. The data were processed using the Daresbury Laue Software Suite to give 2224 independent single reflections (R(merge) = 0.136) in the wavelength range 0.36-1.40 A. The data completeness was 44% at 2.55 A resolution. Phase determination for the data was undertaken using the molecular-replacement method; the top peak was chosen in both the rotation function and the subsequent translation function. This solution agreed well with the molecular-replacement solution achieved independently using monochromatic data. The electron-density map showed reasonably good agreement with the model and the copper site was readily recognizable as it had the highest density. To see if the electron-density map could be improved, ;the doublets in the diffraction data were then deconvoluted. This added 26% data in the region infinity-2d(min) resulting in an improvement in the data completeness to 50% and thus in improved continuity of the electron-density map. The quality of these maps is discussed from the point of view of the suitability of this approach for studying redox-induced structural changes.

Journal Article↗
PubMed1995

Structure of a new azurin from the denitrifying bacterium Alcaligenes xylosoxidans at high resolution.

It has been reported previously that Alcaligenes xylosoxidans (NC1MB 11015) grown under denitrifying conditions produces two azurins instead of the single previously identified azurin [Dodd, Hasnain, Hunter, Abraham, Debenham, Kanzler, Eldridge, Eady, Ambler & Smith (1995). Biochemistry. In the press]. The new azurin, called azurin II, has been crystallized as blue elongated rectangular prisms with the tetragonal space group P4(1)22 and unit-cell parameters a = b = 52.65, c = 100.63 A. X-ray crystallographic data extending to 1.9 A resolution were collected by the Weissenberg method using 200 x 400 mm image plates and synchrotron X-rays of wavelength 0.97 A. The three-dimensional structure of azurin II has been solved by the molecular-replacement method using the structure of azurin from Alcaligenes denitrificans NCTC 8582 with which this new azurin shows a close homology. The quality of the initial map was sufficient to predict a number of sequence differences. The model is currently refined to an R-factor of 18.8% with X-ray data between 8.5 and 1.9 A. The final model of 961 protein atoms, one Cu atom and 50 water molecules has r.m.s. deviations from ideality of 0.009 A for bond lengths and 1.7 degrees for bond angles. The overall structure is similar to that of the azurin from A. denitrificans NCTC 8582. It has a beta-barrel structure with the Cu atom located near the top end of the molecule. The Cu atom is coordinated to Ndelta of His46 and His117 at 2.02 A and to Sgamma of Cys112 at 2.12 A, while the carbonyl O atom of Gly45 and Sdelta atom of Met121 provide the additional interactions at 2.75 and 3.26 A, respectively.

Journal Article↗
PubMed1995

Evidence for two distinct azurins in Alcaligenes xylosoxidans (NCIMB 11015): potential electron donors to nitrite reductase.

We have isolated two type 1 copper-containing proteins (M(r) approximately 13K) from Alcaligenes xylosoxidans (NCIMB 11015) grown under denitrifying conditions. Amino acid sequence analysis of these two proteins shows one to be the previously identified azurin (Ambler, 1971), which we shall call azurin I, and the other to be a related, but previously undescribed, blue copper protein which we show to also be an azurin and propose to call azurin II. Thus, NCIMB 11015 becomes the second system where two distinct azurins are found, the other being Methylomonas J (Ambler & Tobari, 1989). On isoelectric focusing, azurin I migrates very similarly to the previously identified azurin from this organism while azurin II migrates similarly to azurin purified from Alcaligenes denitrificans NCTC 8582. The sequence of azurin II is 33% different than the azurin I sequence but is only 11% different than the azurin from Alcaligenes denitrificans NCTC 8582. Optical spectra for the two proteins are very similar with epsilon mM values of 6.27 and 5.73 mM-1 cm-1 for azurin I and II, respectively, at lambda max approximately 620 nm. The 291 nm shoulder normally ascribed to the hydrophobic nature of tryptophan 48 is clearly observed in azurin I but is missing in azurin II. Amino acid analysis confirms that this tryptophan is missing in azurin II. Azurin I and azurin II show essentially the same redox potential of 305 +/- 10 mV at pH 7.5 and are equally effective electron donors to the purified dissimilatory nitrite reductase of Alc. xylosoxidans in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗
PubMed1995

X-ray absorption studies and homology modeling define the structural features that specify the nature of the copper site in rusticyanin.

Rusticyanin, a blue copper protein, possessing the highest redox potential among this class of proteins and a high stability at acidic pH reveals homology with the C-terminal end of the other single copper containing blue proteins and an interesting homology to parts of the blue copper domain of the multi-copper proteins such as the nitrite reductases. Extended X-ray absorption fine structure (EXAFS) data at pH 2.0 reveal that Cu is ligated to two His and a Cys in the inner coordination sphere, similar to other blue copper centers. Modeling studies suggest that His85 is the ligating histidine from the N-terminal end. Its neighboring residue is a serine rather than the asparagine found in all known blue Cu proteins. The high stability of the copper site may arise in part due to this substitution. The Cu binding site is surrounded by aromatic residues which may provide further protection for the metal in an acidic environment. In addition, the high number of solvent-exposed uncompensated lysine residues is likely to be of functional relevance under low pH conditions. EXAFS data show a very small change (relative to azurin) in the copper site upon reduction, consistent with a more constrained copper center in rusticyanin compared to azurin and a higher redox potential.

Absorptiometry, Photon↗
PubMed1995

Structural and spectroscopic studies of the copper site of stellacyanin.

The structure of the copper site in oxidized and reduced Rhus vernicifera stellacyanin has been studied by X-ray absorption (XAFS) spectroscopy at different pH values. Data for the oxidized protein are consistent with the fourth ligand being an O- or N-donating ligand rather than a cysteine from the disulfide bridge. The fourth ligand is not present in the inner coordination sphere, but makes a more distant interaction 2.7 A from the copper atom. Only minor changes in the details of the Cu(II) coordination occur when the pH is varied. Direct structural information on reduced stellacyanin is provided. Upon reduction, one of the histidine ligands moves away from the copper atom by at least 0.2 A. A low-Z (O or N) scatterer is present approximately 2.4 A from the Cu(I) atom in the protein at low pH, and this ligand is lost at high pH. There is no evidence for an S-donating fourth ligand in the reduced protein. The XAFS results are presented in relation to the spectroscopic and structural information available for some methionine-121 mutants of azurin. The data reveal that there are spectroscopic similarities between stellacyanin and some of the mutant proteins, but distinct structural differences exist that preclude these proteins as suitable models for the copper site of stellacyanin.

Azurin↗
PubMed1995

Quick Fluorescence-EXAFS: an Improved Method for Collection of Conventional XAFS Data, an Improved Method for Collection of Conventional XAFS Data and for Studying Reaction Intermediates in Dilute Systems.

The quick EXAFS (QuEXAFS) technique provides an alternative way of recording X-ray absorption fine-structure (XAFS) data where the scan time is reduced by moving the monochromator at a constant angular speed and recording the data ;on the fly'. Results are presented to show that the use of fluorescence detection with QuEXAFS is eminently suitable for studying reactions in dilute systems such as metalloproteins at a sub-minute time scale. In addition, we show that the fluorescence-QuEXAFS technique can reduce the overall time for normal data collection by some 50% compared with conventional step-by-step scanning EXAFS using the same optical system, thus reducing the total X-ray exposures of the samples. The use of X-rays for studying in situ redox reactions is demonstrated.

Journal Article↗
PubMed1995

The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase.

Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding. X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre. The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu-histidine bond by approximately 0.08 A. Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

Absorptiometry, Photon↗
PubMed1994

Crystallization and preliminary crystallographic data for the azurin mutant End-121 from Pseudomonas aeruginosa.

Pseudomonas aeruginosa azurin has been crystallized from a mutant where residues from Met 121 to Lys128 have been deleted from the protein. The crystals form pale-blue well formed prisms in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 60.79 (5), b = 123.47 (5), c = 187.77 (5) A. The crystals diffract to 3.0 A and there are eight molecules in the asymmetric unit.

Journal Article↗
PubMed1993

X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution.

We demonstrate here the applicability of X-ray scattering for studying molecular conformation of multimeric proteins in solution by using synchrotron radiation to extend the range of data collection to include medium angles (ca. 3-4 degrees). We have been able to define the solution structure of the dissimilatory nitrite reductase of Achromobacter xylosoxidans (AxNiR), an enzyme for which there are conflicting reports as to the nature of its multimeric structure. Quantitative interpretation of the X-ray scattering profile, based on a modeling study using the high-resolution crystal structure data for the nitrite reductase from the related organism Achromobacter cycloclastes (AcNiR), provides a detailed model for the trimeric structure of AxNiR in solution. Sedimentation equilibrium centrifugation gave an M(r) of 103,000, consistent with such a trimeric structure.

Alcaligenes↗
PubMed1993

Asp ligand provides the trigger for closure of transferrin molecules. Direct evidence from X-ray scattering studies of site-specific mutants of the N-terminal half-molecule of human transferrin.

Recent X-ray crystallographic and solution X-ray scattering studies have shown that transferrins (serum transferrin, lactoferrin and ovotransferrin) undergo a major conformational change when iron is incorporated into the molecule. Apo-proteins show a structure with open interdomain clefts which close when iron is bound. The closed conformation has been suggested as an important step in the receptor recognition. Here, we report X-ray solution scattering experiments of the mutated N-terminal fragment of human serum transferrin with Asp63-->Ser (Cys). The data provide the first direct experimental evidence for the existence of a trigger mechanism for the closure of the interdomain cleft and that this trigger mechanism is disrupted by mutation of Asp63, the only ligand of iron from domain I.

Aspartic Acid↗
PubMed1993

Structural characterization of azurin from Pseudomonas aeruginosa and some of its methionine-121 mutants.

Azurin from Pseudomonas aeruginosa and two mutants where the methionine ligand has been mutated have been studied in order to directly investigate the functional and structural significance of this ligand in the blue copper proteins. Reduction potentials, X-ray absorption fine structure (XAFS), electron paramagnetic resonance (EPR), and optical spectra are obtained in an attempt to provide a direct correlation between the spectrochemical properties and the immediate structure of this redox center.

Absorptiometry, Photon↗
PubMed1993

The mechanism of iron uptake by transferrins: the structure of an 18 kDa NII-domain fragment from duck ovotransferrin at 2.3 A resolution.

The molecular structure of an iron-containing 18 kDa fragment of duck ovotransferrin, obtained by proteolysis of the intact protein, has been elucidated by protein crystallographic techniques at 2.3 A resolution. This structure supports a mechanism of iron uptake in the intact protein whereby the binding of the synergistic (bi)carbonate anion is followed by binding of the metal with the lobe in the open configuration. These stages are then followed by domain closure in which the aspartic acid residue plays a further key role, by forming an interdomain hydrogen-bond interaction in addition to serving as a ligand to the iron. This essential dual role is highlighted by model building studies on the C-terminal lobe of a known human variant. In this variant a mutation of a glycine by an arginine residue enables the aspartic acid to form an ion pair and reduce its effectiveness for both metal binding and domain closure. The X-ray structure of the 18 kDa fragment strongly suggests that the histidine residue present at the iron binding site of the intact protein and arising from the second interdomain connecting strand has been removed during the preparative proteolysis.

Journal Article↗
PubMed1993

Metal-induced conformational changes in transferrins.

Recent studies on iron-loaded transferrins have revealed a conformational change upon binding iron due to a domain closure. It has been suggested that the domain closure may be the key for the receptor recognition of the metal loaded transferrin (Grossmann et al., 1992). Small angle X-ray scattering has been used to provide direct structural information on the conformational changes that may take place upon the binding and release of different metals to the transferrins in solution. The data show that In3+ and Cu2+ induce the same domain closure as Fe3+; Al3+ causes a conformational change of somewhat smaller magnitude while Hf4+ does not induce any conformational change. The results are discussed in terms of the molecular recognition of metal loaded transferrin by the receptor.

Metals↗