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K S Wilson

Publications and source records attributed to K S Wilson.

At least 73 records · Page 4Linked to original sources
PubMed1999

A MAD experiment performed at the white line of the iridium LIII absorption edge in lysozyme.

A multiple-wavelength anomalous diffraction (MAD) experiment was performed on an iridium derivative of hen egg-white lysozyme. Diffraction data were measured at five wavelengths on the X31 EMBL beamline on the DORIS III storage ring and at two further wavelengths on the X11 beamline. Four iridium-binding sites were located from the dispersive anomalous differences between two wavelengths at the rising and falling inflection points of the Ir LIII-edge white line using direct methods. All other attempts to determine the heavy-atom positions failed. The results demonstrate an experimental method whereby systematic error in MAD data due to sample absorption can be reduced where a white line is present in the absorption spectrum of a heavy atom.

Animals↗
PubMed1999

Efficient anisotropic refinement of macromolecular structures using FFT.

This paper gives the equations for the use of fast Fourier transformations in individual atomic anisotropic refinement. Restraints on bonded atoms, on the sphericity of each atom and between non-crystallographic symmetry related atoms are described. These have been implemented in the program REFMAC and its performance with several examples is analysed. All the tests show that anisotropic refinement not only reduces the R value and Rfree but also improves the fit to geometric targets, indicating that this parameterization is valuable for improving models derived from experimental data. The computer time taken is comparable to that for isotropic refinements.

Catalase↗
PubMed1999

Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase.

BACKGROUND: The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications - in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. RESULTS: The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 A and 2.0 A resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. CONCLUSIONS: The 2,5B conformation found for the alpha-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside hydrolases.

Bacillus↗
PubMed1999

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.

The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

Catalytic Domain↗
PubMed1999

Detection of antibodies against hepatitis C virus in saliva: a marker of viral replication.

Hepatitis C surveillance has been restricted owing to the lack of a sensitive antibody assay for saliva. The aim of our study was to develop and evaluate a screening assay for hepatitis C antibody in saliva specimens. Serum/saliva pairs were collected from 115 hepatitis C-positive patients. A modified hepatitis C antibody assay for saliva was developed and linked to testing carried out in the diagnostic laboratory. Correlation between the presence of antibody in serum and in saliva was poor (100% vs 85%). However, of 98 patients who were saliva antibody positive, 96 (98%) were also serum hepatitis C RNA positive and two (2%) were serum hepatitis C RNA negative. Hence, the correlation between a positive salivary antibody test and the serum hepatitis C RNA status of intravenous drug users suggests that this test could be used as a surrogate marker for hepatitis C viraemia in epidemiological studies.

Adult↗
PubMed1999

Crystallization and preliminary X-ray diffraction of Trypanosoma cruzi dUTPase.

Crystals of Trypanosoma cruzi dUTPase have been grown. Two different morphologies are observed, depending on the molecular weight of the PEG used as precipitating agent in the mother liquor, both having a hexagonal unit cell with similar dimensions. Complete X-ray diffraction data have been collected to low resolution for one of the forms. The space group is P6322, with unit-cell dimensions a = 134.15, c = 147.05 A. Peaks in the self-rotation function and the solvent content are consistent with two molecules of dUTPase per asymmetric unit.

Animals↗
PubMed1999

The high-resolution structure of DNA-binding protein HU from Bacillus stearothermophilus.

Protein HU is a ubiquitous prokaryotic protein which controls the architecture of genomic DNA. It binds DNA non-specifically and promotes the bending and supercoiling of the double helical structure. HU is involved in many DNA-associated cellular processes, including replication, transcription and the packaging of DNA into chromosome-like structures. Originally determined at medium resolution, the crystal structure of HU has now been refined at 2.0 A resolution. The high-resolution structure shows that the dimeric molecule is essentially a compact platform for two flexible and basic arms which wrap around the DNA molecule. To maximize the protein's stability, non-secondary structural regions are reduced to a minimum, there is an extensive aromatic hydrophobic core and several salt bridges and hydrogen-bonded water molecules knit together crucial regions. Based on the original medium-resolution structure of HU, several proposals were made concerning the structural basis of HU's ability to bind, bend and supercoil DNA. Each of these proposals is fully supported by the high-resolution structure. Most notably, the surfaces of the molecule which appear to mediate protein-DNA and protein-protein interactions have the ideal shapes and physicochemical properties to perform these functions.

Bacterial Proteins↗
PubMed1999

Crystallization of a precursor penicillin acylase from Escherichia coli.

The crystallization of a mutant precursor penicillin acylase [penicillin amidohydrolase (amidase) E.C. 3.5.1.11] from Escherichia coli W (ATCC 11105) using the hanging-drop method is reported. The crystals are in space group P1 with unit-cell parameters a = 51.04, b = 63.58, c = 71.17 A, alpha = 103.0, beta = 110.6, gamma = 105.3 degrees, with one molecule in the asymmetric unit, and diffract to 1. 8 A using synchrotron radiation.

Bacterial Proteins↗
PubMed1999

Crystallization and preliminary X-ray crystallographic analysis of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5.

Crystals of the catalytic core domain of a Trichoderma reesei beta-mannanase belonging to glycoside hydrolase family 5 have been grown by the sitting-drop method at room temperature using ammonium sulfate as precipitant. The crystals grow as thin colourless plates and belong to space group P21, with unit-cell parameters a = 50.0, b = 54.3, c = 60.2 A, beta = 111.3 degrees, and have a single monomer of mannanase in the asymmetric unit. Native data to 2.0 A resolution have been collected at room temperature using synchrotron radiation. Data for a platinum derivative have been collected to 1.65 A at 110 K in a very short time at the CCLRC Daresbury synchrotron source, using a charge-coupled device (CCD) as detector.

Catalysis↗
PubMed1998

Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1.

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.

Asparagine↗
PubMed1998

Structural parameters for proteins derived from the atomic resolution (1.09 A) structure of a designed variant of the ColE1 ROP protein.

The crystal structure of a designed variant of the ColE1 repressor of primer (ROP) protein has been refined with SHELXL93 to a resolution of 1.09 A. The final model with 510 non-H protein atoms, 576 H atoms in calculated positions and 114 water molecules converged to a standard R factor of 10% using unrestrained blocked full-matrix refinement. For all non-H atoms six-parameter anisotropic thermal parameters have been refined. The majority of atomic vibrations have a preferred orientation which is approximately perpendicular to the bundle axis; analysis with the TLS method [Schomaker & Trueblood (1968). Acta Cryst. B24, 63-77] showed a relatively good agreement between the individual atomic displacements and a rigid-body motion of the protein. Disordered residues with multiple conformations form clusters on the surface of the protein; six C-terminal residues have been omitted from the refined model due to disorder. Part of the solvent structure forms pentagonal or hexagonal clusters which bridge neighbouring protein molecules. Some water molecules are also conserved in wild-type ROP. The unrestrained blocked full-matrix least-squares refinement yielded reliable estimates of the standard deviations of the refined parameters. Comparison of these parameters with the stereochemical restraints used in various protein refinement programs showed statistically significant differences. These restraints should be adapted to the refinement of macromolecules by taking into account parameters determined from atomic resolution protein structures.

Anisotropy↗
PubMed1998

Expression, crystallization and preliminary X-ray analysis of the E2 transactivation domain from papillomavirus type 16.

The N-terminal transactivation domain of the E2 protein from human papillomavirus type 16 has been crystallized by vapour diffusion. Crystals belong to the space group P3121 (or P3221) with unit-cell dimensions a = b = 54.3, c = 155.5 A. There is one molecule per asymmetric unit with a solvent content of 55%. Crystals diffract to at least 2.5 A resolution and complete X-ray data to 3.4 A have been collected on a conventional laboratory source. This 201 amino-acid domain of the E2 protein has been shown to interact functionally with both the HPV E1 protein and at least three cellular transcription factors, to fulfil its role in the control of viral transcription and replication. A knowledge of the structural basis of these multiple interactions should lead to a fuller understanding of the mechanism of action of this key regulator of the HPV life cycle.

Cloning, Molecular↗
PubMed1998

Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg.

The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (= sigma absolute value [(absolute value Fo) - (absolute value Fc)] / sigma (absolute value of Fo) of 15.1% using X-ray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.

Amino Acid Sequence↗
PubMed1998

The refined structure of dUTPase from Escherichia coli.

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6. 1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis. A crystal of the recombinant E. coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 1.9 A resolution, in space group R3, a = b = 86.62, c = 62.23 A. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 A MIRAS phases differed from the final set by 40 degrees on average and produced an excellent map which was easy to interpret. The model contains 132 water molecules and refined to an R value of 13.7%. 136 residues have clear electron density out of 152 expected from the gene sequence. The 16 C-terminal residues are presumably disordered in the crystal lattice. The monomer is a 'jelly-roll' type, containing mostly beta-sheet and only one short helix. The molecule is a tight trimer. A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its beta-sheet. Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the beta-sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.

Amino Acid Sequence↗
PubMed1998

Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 A resolution.

The yeast Saccharomycopsis fibuligera produces a glucoamylase which belongs to sequence family 15 of glycosyl hydrolases. The structure of the non-glycosyl-ated recombinant enzyme has been determined by molecular replacement and refined against 1.7 A resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-dimensional structure of a yeast family 15 glucoamylase. The refinement from the initial molecular-replacement model was not straightforward. It involved the use of an unrestrained automated refinement procedure (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alpha-helices, 12 of which form an (alpha/alpha)6 barrel. It contains a single catalytic domain but no starch-binding domain. The fold of the molecule and the active site are compared to the known structure of the catalytic domain of a fungal family 15 glucoamylase and are shown to be closely similar. The active- and specificity-site residues are especially highly conserved. The model of the acarbose inhibitor from the analysis of the fungal enzyme fits tightly into the present structure. The active-site topology is a pocket and hydrolysis proceeds with inversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.

Amino Acid Sequence↗
PubMed1998

Recognition of RNase Sa by the inhibitor barstar: structure of the complex at 1.7 A resolution.

We report the 1.7 A resolution structure of RNase Sa complexed with the polypeptide inhibitor barstar. The crystals are in the hexagonal space group P65 with unit-cell dimensions a = b = 56.9, c = 135.8 A and the asymmetric unit contains one molecule of the complex. RNase Sa is an extracellular microbial ribonuclease produced by Streptomyces aureofaciens. Barstar is the natural inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens. It inhibits RNase Sa and barnase in a similar manner by steric blocking of the active site. The structure of RNase Sa is very similar to that observed in crystals of the native enzyme and its complexes with nucleotides. Barstar retains the structure found in its complex with barnase. The accessible surface area of protein buried in the complex is about 300 A2 smaller and there are fewer hydrogen bonds in the enzyme-inhibitor interface in RNase Sa-barstar than in barnase-barstar, providing an explanation of the reduced binding affinity in the former. Previous studies of barstar complexes have used mutants of the inhibitor and this is the first structure which includes wild-type barstar.

Amino Acid Sequence↗
PubMed1998

Refinement of triclinic hen egg-white lysozyme at atomic resolution.

X-ray diffraction data have been collected at both low (120 K) and room temperature from triclinic crystals of hen egg-white lysozyme to 0.925 and 0.950 A resolution, respectively, using synchrotron radiation. Data from one crystal were sufficient for the low-temperature study, whereas three crystals were required at room temperature. Refinement was carried out using the programs PROLSQ, ARP and SHELXL to give final conventional R factors of 8.98 and 10.48% for data with F > 4sigma(F) for the low- and room-temperature structures, respectively. The estimated r.m.s. coordinate error is 0.032 A for protein atoms, 0.050 A for all atoms in the low-temperature study, and 0.038 A for protein atoms and 0.049 A for all atoms in the room-temperature case, as estimated from inversion of the blocked least-squares matrix. The low-temperature study revealed that the side chains of 24 amino acids had multiple conformations. A total of 250 waters, six nitrate ions and three acetate ions, two of which were modelled with alternate orientations were located in the electron-density maps. Three sections of the main chain were modelled in alternate conformations. The room-temperature study produced a model with multiple conformations for eight side chains and a total of 139 water molecules, six nitrate but no acetate ions. The occupancies of the water molecules were refined in both structures and this step was shown to be meaningful when assessed by use of the free R factor. A detailed description and comparison of the structures is made with reference to the previously reported structure refined at 2.0 A resolution.

Animals↗