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G L Taylor

Publications and source records attributed to G L Taylor.

At least 19 recordsLinked to original sources
PubMed2002

Structure of Alba: an archaeal chromatin protein modulated by acetylation.

Eukaryotic DNA is packaged into nucleosomes that regulate the accessibility of the genome to replication, transcription and repair factors. Chromatin accessibility is controlled by histone modifications including acetylation and methylation. Archaea possess eukary otic-like machineries for DNA replication, transcription and information processing. The conserved archaeal DNA binding protein Alba (formerly Sso10b) interacts with the silencing protein Sir2, which regulates Alba's DNA binding affinity by deacetylation of a lysine residue. We present the crystal structure of Alba from Sulfolobus solfataricus at 2.6 A resolution (PDB code 1h0x). The fold is reminiscent of the N-terminal DNA binding domain of DNase I and the C-terminal domain of initiation factor IF3. The Alba dimer has two extended beta-hairpins flanking a central body containing the acetylated lysine, Lys16, suggesting three main points of contact with the DNA. Fluorescence, calorimetry and electrophoresis data suggest a final binding stoichiometry of approximately 5 bp DNA per Alba dimer. We present a model for the Alba-DNA interaction consistent with the available structural, biophysical and electron microscopy data.

Acetylation↗
PubMed2001

Tiny TIM: a small, tetrameric, hyperthermostable triosephosphate isomerase.

Comparative structural studies on proteins derived from organisms with growth optima ranging from 15 to 100 degrees C are beginning to shed light on the mechanisms of protein thermoadaptation. One means of sustaining hyperthermostability is for proteins to exist in higher oligomeric forms than their mesophilic homologues. Triosephosphate isomerase (TIM) is one of the most studied enzymes, whose fold represents one of nature's most common protein architectures. Most TIMs are dimers of approximately 250 amino acid residues per monomer. Here, we report the 2.7 A resolution crystal structure of the extremely thermostable TIM from Pyrococcus woesei, a hyperthermophilic archaeon growing optimally at 100 degrees C, representing the first archaeal TIM structure. P. woesei TIM exists as a tetramer comprising monomers of only 228 amino acid residues. Structural comparisons with other less thermostable TIMs show that although the central beta-barrel is largely conserved, severe pruning of several helices and truncation of some loops give rise to a much more compact monomer in the small hyperthermophilic TIM. The classical TIM dimer formation is conserved in P. woesei TIM. The extreme thermostability of PwTIM appears to be achieved by the creation of a compact tetramer where two classical TIM dimers interact via an extensive hydrophobic interface. The tetramer is formed through largely hydrophobic interactions between some of the pruned helical regions. The equivalent helical regions in less thermostable dimeric TIMs represent regions of high average temperature factor. The PwTIM seems to have removed these regions of potential instability in the formation of the tetramer. This study of PwTIM provides further support for the role of higher oligomerisation states in extreme thermal stabilisation.

Amino Acid Sequence↗
PubMed2000

Crystallization of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein.

The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was isolated by cleaving HN (cHN) from reconstituted virosome with chymotrypsin. N-terminal sequence analysis of the purified cHN showed that chymotrypsin cleavage had occurred at amino acid 123, freeing the C-terminal 454 amino acids. The purified cHN retained its neuraminidase and receptor binding activities and reacted with specific monoclonal antibodies, showing that the isolated cHN was biologically and antigenically functional. The crystals of the cHN were obtained in acetate buffer (pH 4.6) containing polyethylene glycol 3350 and ammonium sulfate and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimension of approximately a = 72 A, b = 78 A, and c = 198 A. Crystals of cHN grown in the presence of sialic acid (Neu5Ac) were grown in HEPES buffer (pH 6.2) containing polyethylene glycol 3350 and belong to the hexagonal space groups P6(1) or P6(5) with unit cell dimensions of a = b = 137.5 A and c = 116.6A. The orthorhombic crystals produced in this study diffract X rays to at least 2.0-A resolution, thereby setting the stage for the solution of the three-dimensional structure of the HN glycoprotein of a paramyxovirus.

Amino Acid Sequence↗
PubMed2000

Age dependence of heat stress mediated cardioprotection.

BACKGROUND: To study the influence of age on heat stress cardioprotection, functional recovery, nucleotide concentrations, and heat stress protein 70 (Hsp70) levels were compared in the heat stressed (HS) and control (C) hearts at different ages, in a protocol mimicking donor heart preservation for transplantation. METHODS: Control and heat stressed (24 hours before experiment) rat hearts were divided into three age groups: (I) 1 month, (Y) 4 months, and (M) 16 months (n = 6). Left ventricle balloon catheter was used to determine systolic and end-diastolic pressure/volume relations before and after 4 hours of cardioplegic arrest at 4 degrees C. Another identical set of isolated hearts underwent 5 minutes of normoxic perfusion to obtain preischemic Hsp70 content and metabolite concentrations. RESULTS: The postischemic recovery was highest in group HS-Y as compared to C-Y, HS-I, C-I, HS-M, and C-M. There were no differences in preischemic adenine nucleotides or creatine metabolite concentrations between the three age groups. In contrast, the nicotinamide adenine dinucleotide (oxidized form) (NAD+) and nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP+) concentrations were significantly raised in group HS-Y. Hsp70 content was increased in all HS groups with no difference between the age groups. CONCLUSIONS: Improved postischemic functional recovery after cardioplegic arrest was observed in the young adult HS hearts. This was associated with highest NAD+ and NADP+ concentrations and did not correlate with increased Hsp70 content.

Aging↗
PubMed2000

Preliminary crystallographic studies of an extremely thermostable KDG aldolase from Sulfolobus solfataricus.

Crystals have been grown of 2-keto-3-deoxygluconate aldolase (KDG aldolase) from the hyperthermophilic archaeon Sulfolobus solfataricus that diffract to 2.2 A resolution. The enzyme catalyses the reversible aldol cleavage of 2-keto-3-dexoygluconate to pyruvate and glyceraldehyde, the third step of a modified non-phosphorylated Entner-Doudoroff pathway of glucose oxidation. S. solfataricus grows optimally at 353 K and the enzyme itself has a half-life of 2.5 h at 373 K. Knowledge of the crystal structure of KDG aldolase will further understanding of the basis of protein hyperthermostability and create a target for site-directed mutagenesis of active-site residues, with the aim of altering substrate specificity. Three crystal forms have been obtained: orthorhombic crystals of space group P2(1)2(1)2(1), which diffract to beyond 2.15 A, monoclinic crystals of space group C2, which diffract to 2.2 A, and cubic crystals of space group P4(2)32, which diffract to 3.4 A.

Aldehyde-Lyases↗
PubMed1998

Preliminary crystallographic studies of triosephosphate isomerase (TIM) from the hyperthermophilic Archaeon Pyrococcus woesei.

Recombinant triosephosphate isomerase (TIM) from a hyperthermophilic Archaeon, Pyrococcus woesei, has been crystallized. Three crystal forms have been obtained: monoclinic, orthorhombic and hexagonal. The monoclinic crystals belong to space group P21 with cell dimensions a = 79.1, b = 89.2, c = 145.4 A and beta = 92.8 degrees, and diffract to at least 2.6 A. The orthorhombic crystals belong to space group P21212 with a = 89.4, b = 155.9, c = 79.5 A, and diffract to 2.9 A. Diffraction from the hexagonal form showed extensive disorder. The monoclinic form contains two tetramers in the asymmetric unit, which are in the same orientation but related by a pseudo-centering. The orthorhombic form contains one tetramer in the asymmetric unit which is in approximately the same orientation as in the monoclinic form. Knowledge of the structure of this hyperthermostable TIM, which is tetrameric in contrast to dimeric forms previously observed, will add to our understanding of protein thermostability.

Bacterial Proteins↗
PubMed1998

Preliminary crystallographic studies of citrate synthase from an Antarctic psychrotolerant bacterium.

Recombinant citrate synthase from a psychrotolerant bacterium, DS2-3R, recently isolated in Antarctica, has been crystallized. The crystals belong to space group P6122 or P6522, with cell dimensions a = b = 70.8, c = 307.8 A. Diffraction data collected on a synchrotron from a cryoprotected crystal extends to at least 2.0 A. Knowledge of the structure of this enzyme will add to the understanding of cold activity and thermolability, and will be of biotechnological interest. Previously, the structure of citrate synthase from Archaea inhabiting environments at 328 and 373 K, has been reported. This present study will extend our understanding of the structural integrity and activity of proteins at the temperature extremes of life.

Adaptation, Physiological↗
PubMed1998

Structural adaptations of the cold-active citrate synthase from an Antarctic bacterium.

BACKGROUND: The structural basis of adaptation of enzymes to low temperature is poorly understood. Dimeric citrate synthase has been used as a model enzyme to study the structural basis of thermostability, the structure of the enzyme from organisms living in habitats at 55 degrees C and 100 degrees C having previously been determined. Here the study is extended to include a citrate synthase from an Antarctic bacterium, allowing us to explore the structural basis of cold activity and thermostability across the whole temperature range over which life is known to exit. RESULTS: We report here the first crystal structure of a cold-active enzyme, citrate synthase, isolated from an Antarctic bacterium, at a resolution of 2.09 A. In comparison with the same enzyme from a hyperthermophilic host, the cold-active enzyme has a much more accessible active site, an unusual electrostatic potential distribution and an increased relative flexibility of the small domain compared to the large domain. Several other features of the cold-active enzyme were also identified: reduced subunit interface interactions with no intersubunit ion-pair networks; loops of increased length carrying more charge and fewer proline residues; an increase in solvent-exposed hydrophobic residues; and an increase in intramolecular ion pairs. CONCLUSIONS: Enzymes from organisms living at the temperature extremes of life need to avoid hot or cold denaturation yet maintain sufficient structural integrity to allow catalytic efficiency. For hyperthermophiles, thermal denaturation of the citrate synthase dimer appears to be resisted by complex networks of ion pairs at the dimer interface, a feature common to other hyperthermophilic proteins. For the cold-active citrate synthase, cold denaturation appears to be resisted by an increase in intramolecular ion pairs compared to the hyperthermophilic enzyme. Catalytic efficiency of the cold-active enzyme appears to be achieved by a more accessible active site and by an increase in the relative flexibility of the small domain compared to the large domain.

Amino Acid Sequence↗
PubMed1997

The crystal structure of citrate synthase from the hyperthermophilic archaeon pyrococcus furiosus at 1.9 A resolution,.

The crystal structure of the closed form of citrate synthase, with citrate and CoA bound, from the hyperthermophilic Archaeon Pyrococcus furiosus has been determined to 1.9 A. This has allowed direct structural comparisons between the same enzyme from organisms growing optimally at 37 degrees C (pig), 55 degrees C (Thermoplasma acidophilum) and now 100 degrees C (Pyrococcus furiosus). The three enzymes are homodimers and share a similar overall fold, with the dimer interface comprising primarily an eight alpha-helical sandwich of four antiparallel pairs of helices. The active sites show similar modes of substrate binding; moreover, the structural equivalence of the amino acid residues implicated in catalysis implies that the mechanism proceeds via the same acid-base catalytic process. Given the overall structural and mechanistic similarities, it has been possible to make detailed structural comparisons between the three citrate synthases, and a number of differences can be identified in passing from the mesophilic to thermophilic to hyperthermophilic citrate synthases. The most significant of these are an increased compactness of the enzyme, a more intimate association of the subunits, an increase in intersubunit ion pairs, and a reduction in thermolabile residues. Compactness is achieved by the shortening of a number of loops, an increase in the number of atoms buried from solvent, an optimized packing of side chains in the interior, and an absence of cavities. The intimate subunit association in the dimeric P. furiosus enzyme is achieved by greater complementarity of the monomers and by the C-terminal region of each monomer folding over the surface of the other monomer, in contrast to the pig enzyme where the C-terminus has a very different fold. The increased number of intersubunit ion pairs is accompanied by an increase in the number involved in networks. Interestingly, all loop regions in the P. furiosus enzyme either are shorter or contain additional ion pairs compared with the pig enzyme. The possible relevance of these structural features to enzyme hyperthermostability is discussed.

Amino Acid Sequence↗
PubMed1997

Identification of a sialidase encoded in the human major histocompatibility complex.

Mammalian sialidases are important in modulating the sialic acid content of cell-surface and intracellular glycoproteins. However, the full extent of this enzyme family and the physical and biochemical properties of its individual members are unclear. We have identified a novel gene, G9, in the human major histocompatibility complex (MHC), that encodes a 415-amino acid protein sharing 21-28% sequence identity with the bacterial sialidases and containing three copies of the Asp-block motif characteristic of these enzymes. The level of sequence identity between human G9 and a cytosolic sialidase identified in rat and hamster (28-29%) is much less than would be expected for analogous proteins in these species, suggesting that G9 is distinct from the cytosolic enzyme. Expression of G9 in insect cells has confirmed that it encodes a sialidase, which shows optimal activity at pH 4.6, but appears to have limited substrate specificity. The G9 protein carries an N-terminal signal sequence and immunofluorescence staining of COS7 cells expressing recombinant G9 shows localization of this sialidase exclusively to the endoplasmic reticulum. The location of the G9 gene, within the human MHC, corresponds to that of the murine Neu-1 locus, suggesting that these are analogous genes. One of the functions attributed to Neu-1 is the up-regulation of sialidase activity during T cell activation.

Amino Acid Sequence↗
PubMed1996

The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution.

The structure of Salmonella typhimurium LT2 neuraminidase (STNA) is reported here to a resolution of 1.6 angstroms together with the structures of three complexes of STNA with different inhibitors. The first is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en or DANA), the second and third are phosphonate derivatives of N-acetyl-neuraminic acid (NANA) which have phosphonate groups at the C2 position equatorial (ePANA) and axial (aPANA) to the plane of the sugar ring. The complex structures are at resolutions of 1.6 angstroms, 1.6 angstroms and 1.9 angstroms, respectively. These analyses show the STNA active site to be topologically inflexible and the interactions to be dominated by the arginine triad, with the pyranose rings of the inhibitors undergoing distortion to occupy the space available. Solvent structure differs only around the third phosphonate oxygen, which attracts a potassium ion. The STNA structure is topologically identical to the previously reported influenza virus neuraminidase structures, although very different in detail; the root-mean-square (r.m.s) deviation for 210 C alpha positions considered equivalent is 2.28 angstroms (out of a total of 390 residues in influenza and 381 in STNA). The active site residues are more highly conserved, in that both the viral and bacterial structures contain an arginine triad, a hydrophobic pocket, a tyrosine and glutamic acid residue at the base of the site and a potential proton-donating aspartic acid. However, differences in binding to O4 and to the glycerol side-chain may reflect the different kinetics employed by the two enzymes.

Binding Sites↗
PubMed1996

Structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases.

Quinone oxidoreductase, zeta-crystallin, glucose dehydrogenase, and alcohol dehydrogenase belong to a superfamily of medium-chain dehydrogenase/reductases. The crystal structures of Escherichia coli quinone oxidoreductase (QOR) and Thermoplasma acidophilum glucose dehydrogenase have recently been determined and are compared here with the well-known structure of horse liver alcohol dehydrogenase. A structurally based comparison of these three enzymes confirms that they possess extensive overall structural homology despite low sequence identity. The most significant difference is the absence of the catalytic and structural zinc ions in QOR. A multiple structure-based sequence alignment has been constructed for the three enzymes and extended to include zeta-crystallin, an eye lens structural protein with quinone oxidoreductase activity and high sequence identity to E. coli quinone oxidoreductase. Residues which are important for catalysis have been altered and the functions and activities of the enzymes have diverged, illustrating a classic example of divergent evolution among a superfamily of enzymes.

Alcohol Dehydrogenase↗
PubMed1995

Engineering thermostability: lessons from thermophilic proteins.

As several groups begin to tap the rich pickings found in the Archaea--a vast kingdom that stretches the concept of life as we know it--the structures of proteins from hyperthermophiles are being elucidated. Certain features are beginning to emerge, such as compactness and hydrophobic clustering, but the ability to engineer these features into temperature-intolerant proteins is still some way off.

Amino Acids↗
PubMed1994

The crystal structure of citrate synthase from the thermophilic archaeon, Thermoplasma acidophilum.

BACKGROUND: The Archaea constitute a phylogenetically distinct, evolutionary domain and comprise organisms that live under environmental extremes of temperature, salinity and/or anaerobicity. Different members of the thermophilic Archaea tolerate temperatures in the range 55-110 degrees C, and the comparison of the structures of their enzymes with the structurally homogolous enzymes of mesophilic organisms (optimum growth temperature range 15-45 degrees C) may provide important information on the structural basis of protein thermostability. We have chosen citrate synthase, the first enzyme of the citric acid cycle, as a model enzyme for such studies. RESULTS: We have determined the crystal structure of Thermoplasma acidophilum citrate synthase to 2.5 A and have compared it with the citrate synthase from pig heart, with which it shares a high degree of structural homology, but little sequence identity (20%). CONCLUSIONS: The three-dimensional structural comparison of thermophilic and mesophilic citrate synthases has permitted catalytic and substrate-binding residues to be tentatively assigned in the archaeal, thermophilic enzyme, and has identified structural features that may be responsible for its thermostability.

Amino Acid Sequence↗
PubMed1994

The crystal structure of glucose dehydrogenase from Thermoplasma acidophilum.

BACKGROUND: The archaea are a group of organisms distinct from bacteria and eukaryotes. Structures of proteins from archaea are of interest because they function in extreme environments and because structural studies may reveal evolutionary relationships between proteins. The enzyme glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum is of additional interest because it is involved in an unusual pathway of sugar metabolism. RESULTS: We have determined the crystal structure of this glucose dehydrogenase to 2.9 A resolution. The monomer comprises a central nucleotide-binding domain, common to other nucleotide-binding dehydrogenases, flanked by the catalytic domain. Unexpectedly, we observed significant structural homology between the catalytic domain of horse liver alcohol dehydrogenase and T. acidophilum glucose dehydrogenase. CONCLUSIONS: The structural homology between glucose dehydrogenase and alcohol dehydrogenase suggests an evolutionary relationship between these enzymes. The quaternary structure of glucose dehydrogenase may provide a model for other tetrameric alcohol/polyol dehydrogenases. The predicted mode of nucleotide binding provides a plausible explanation for the observed dual-cofactor specificity, the molecular basis of which can be tested by site-directed mutagenesis.

Alcohol Dehydrogenase↗