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See detailCrystal Structure of the Lysozyme from Bacteriophage Lambda and its Relationship with V and C-type Lysozymes
Evrard, Christine ULg; Fastrez, Jacques; Declercq, Jean-Paul

in Journal of Molecular Biology (1998), 276

Like other lysozymes, the bacteriophage lambda lysozyme is involved in the digestion of bacterial walls. This enzyme is remarkable in that its mechanism of action is different from the classical lysozyme ... [more ▼]

Like other lysozymes, the bacteriophage lambda lysozyme is involved in the digestion of bacterial walls. This enzyme is remarkable in that its mechanism of action is different from the classical lysozyme's mechanism. From the point of view of protein evolution, it shows features of lysozymes from different classes. The crystal structure of the enzyme in which all tryptophan residues have been replaced by aza-tryptophan has been solved by X-ray crystallography at 2.3 Å using a combination of multiple isomorphous replacement, non-crystallographic symmetry averaging and density modification techniques. There are three molecules in the asymmetric unit. The characteristic structural elements of lysozymes are conserved: each molecule is organized in two domains connected by a helix and the essential catalytic residue (Glu19) is located in the depth of a cleft between the two domains. This cleft shows an open conformation in two of the independent molecules, while access to the cavity is much more restricted in the last one. A structural alignment with T4 lysozyme and hen egg white lysozyme allows us to superpose about 60 Cα atoms with a rms distance close to 2 Å. The best alignments concern the helix preceding the catalytic residue, some parts of the beta sheets and the helix joining the two domains. The results of sequence alignments with the V and C lysozymes, in which weak local similarities had been detected, are compared with the structural results. [less ▲]

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See detailThe origin of the alpha-domain intermediate in the folding of hen lysozyme
Matagne, André ULg; Chung, E. W.; Ball, L. J. et al

in Journal of Molecular Biology (1998), 277(5), 997-1005

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to ... [more ▼]

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in conjunction with quenched-flow hydrogen exchange labelling, monitored by electrospray ionization mass spectrometry, to compare the refolding kinetics of hen egg-white lysozyme at 20 degrees C and 50 degrees C. At 50 degrees C there is clear evidence for distinct fast and slow refolding populations, as observed at 20 degrees C, although folding occurs significantly more rapidly. The folding process is, however, substantially more cooperative at the higher temperature. In particular, the transient intermediate on the major refolding pathway at 20 degrees C, having persistent native-like structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labelling at 50 degrees C. Ln addition, the characteristic maximum in negative ellipticity and the minimum in fluorescence intensity observed in far UV CD and intrinsic fluorescence experiments at 20 degrees C, respectively, are not seen at 50 degrees C. Addition of 2 M NaCl to the refolding buffer at 50 degrees C, however, regenerates both the hydrogen exchange and optical properties associated with the alpha-domain intermediate but has no significant effect on the overall refolding kinetics. Together with previous findings, these results indicate that non-native interactions within the alpha-domain intermediate are directly responsible for the unusual optical properties observed during refolding, and that this intermediate accumulates as a consequence of its intrinsic stability in a folding process where the formation of stable structure in the beta-domain constitutes the rate-limiting step for the majority of molecules. (C) 1998 Academic Press Limited. [less ▲]

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See detailThe 118-135 Peptide Lot The Human Prion Protein Forms Amyloid Fibrils And Induces Liposome Fusion
Pillot, T.; Lins, Laurence ULg; Goethals, M. et al

in Journal of Molecular Biology (1997), 274(3), 381-93

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and ... [more ▼]

The prion protein (PrPC) is a glycoprotein of unknown function normally found at the surface of neurons and of glial cells. It is involved in diseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakob disease in the human, where PrPC is converted into an altered form (termed PrPSc). PrPSc is highly resistant towards proteolytic degradation and accumulates in the central nervous system of affected individuals. By analogy with the pathological events occuring during the development of Alzheimer's disease, controverses still exist regarding the relationship between amyloidogenesis, prion aggregation and neuronal loss. To unravel the mechanism of PrP neurotoxicity and understand the interaction of PrP with cellular membranes, a series of natural and variant peptides spanning residues 118 to 135 of PrP was synthesized. The potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. According to computer modeling calculations, the 120 to 133 domain of PrP is predicted to be a tilted lipid-associating peptide, and to insert in a oblique way into a lipid bilayer through its N-terminal end. In addition to amyloidogenic properties exhibited in vitro by these peptides, peptide-induced vesicle fusion was demonstrated by several techniques, including lipid- and core-mixing assays. Elongation of the 120 to 133 peptide towards the N- and C-terminal ends of the PrP sequence showed that the 118 to 135 PrP peptide has maximal fusogenic properties, while the variant peptides had no effect. Due to their high hydrophobicity, all peptides tested were able to interact with liposomes to induce leakage of encapsulated calcein. We demonstrate also that the propensity of the peptides to fold as an alpha-helix increases their fusogenic activity, thus accounting for the maximal fusogenic activity of the most stable helix at residues 118 to 135. These data suggest that, by analogy with the C-terminal domain of the beta-amyloid peptide, the fusogenic properties exhibited by the prion peptides might contribute to the neurotoxicity of these peptides by destabilizing cellular membranes. [less ▲]

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See detailFast and slow tracks in lysozyme folding: Insight into the role of domains in the folding process
Matagne, André ULg; Radford, S. E.; Dobson, C. M.

in Journal of Molecular Biology (1997), 267(5), 1068-1074

The folding of lysozyme involves parallel events in which hydrogen exchange kinetics indicate the development of persistent structure at very different rates. We have monitored directly the kinetics of ... [more ▼]

The folding of lysozyme involves parallel events in which hydrogen exchange kinetics indicate the development of persistent structure at very different rates. We have monitored directly the kinetics of formation of the native molecule by the binding of a fluorescently labelled inhibitor, MeU-diNAG (4-methylumbelliferyl-N,N'-diacetyl-beta-D-chitobioside). The data show that native character monitored in this way also develops with different timescales. Although the rate determining step on the slow pathway (similar to 75% of molecules at pH 5.5, 20 degrees C) can be attributed to the need to reorganise structure formed early in the folding process, the data indicate that the rate determining step on the fast track (involving similar to 25% of molecules) involves the docking of the two constituent domains of the protein. In the fast folding track the data are consistent with a model in which each domain forms persistent structure prier to their docking in a locally cooperative manner on a timescale comparable to the folding of small single domain proteins. (C) 1997 Academic Press Limited. [less ▲]

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See detailThree hTIM mutants that provide new insights on why TIM is a dimer
Mainfroid, Véronique; Terpstra, Peter; Beauregard, Marc et al

in Journal of Molecular Biology (1996), 257(2), 441-56

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in ... [more ▼]

Human triosephosphate isomerase (hTIM), a dimeric enzyme, was altered by site-directed mutagenesis in order to determine whether it can be dissociated into monomers. Two hTIM mutants were produced, in which a glutamine residue was substituted for either Met14 or Arg98, both of which are interface residuces. These substitutions strongly interfere with TIM subunit association, since these mutant TIMs appear to exist as compact monomers in dynamic equilibrium with dimers. In kinetic studies, the M14Q mutant exhibits significant catalytic activity, while the R98Q enzyme is inactive. The M14Q enzyme is nevertheless much less active than unmutated hTIM. Moreover, its specific activity is concentration dependent, suggesting a dissociation process in which the monomers are inactive. In order to determine the conformational stability of the wild-type and mutant hTIMs, unfolding of all three enzymes was monitored by circular dichroism and tryptophan fluorescence spectroscopy. In each case, protein stability is concentration dependent, and the unfolding reaction is compatible with a two-state model involving the native dimer and unfolded monomers. The conformational stability of hTIM, as estimated according to this model, is 19.3 (+/-0.4) kcal/mol. The M14Q and R98Q replacements significantly reduce enzyme stability, since the free energies of unfolding are 13.8 and 13.5 (+/- 0.3) kcal/mol respectively, for the mutants, A third mutant, in which the M14Q and R98Q replacements are cumulated, behaves like a monomer. The stability of this mutant is not concentration-dependent, and the unfolding reaction is assigned to a transition from a folded monomer to an unfolded monomer. The conformational stability of this double mutant is estimated 2.5 (+/-0.1) kcal/mol. All these data combined suggest that TIM monomers are thermodynamically unstable. This might explain why TIM occurs only as a dimer. [less ▲]

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See detailCrystallization and X-ray diffraction study of the Streptomyces K15 penicillin-binding DD-transpeptidase.
Englebert, S.; Charlier, Paulette ULg; Fonze, E. et al

in Journal of Molecular Biology (1994), 241(2), 295-7

The 262 amino acid residue long DD-transpeptidase/penicillin-binding protein of Streptomyces K15 has been crystallized at room temperature by using the hanging drop vapour diffusion technique. The ... [more ▼]

The 262 amino acid residue long DD-transpeptidase/penicillin-binding protein of Streptomyces K15 has been crystallized at room temperature by using the hanging drop vapour diffusion technique. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 46.4 A, b = 54.1 A and c = 108.3 A. They contain one protein molecule per asymmetric unit and diffract to about 1.9 A. X-ray data have been collected to 2.0 A from a native crystal. The previously published amino acid sequence of the protein has been corrected at positions 71, 72, 113, 114 and 156. [less ▲]

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See detailCrystallization of a genetically engineered water-soluble primary penicillin target enzyme. The high molecular mass PBP2x of Streptococcus pneumoniae.
Charlier, Paulette ULg; Buisson, G.; Dideberg, O. et al

in Journal of Molecular Biology (1993), 232(3), 1007-9

A genetically engineered water-soluble derivative of PBP2x of Streptococcus pneumoniae has been produced, purified and crystallized in a form suitable for X-ray diffraction analysis. The best crystals ... [more ▼]

A genetically engineered water-soluble derivative of PBP2x of Streptococcus pneumoniae has been produced, purified and crystallized in a form suitable for X-ray diffraction analysis. The best crystals have been grown at 15 degrees C, from solutions containing 8% polyethylene glycol 10,000 at pH values ranging from 3.9 to 6.0. These crystals diffract to a resolution of 3.5 A and have a space group P6(1)22 (or enantiomorph) with unit cell dimensions of a = b = 162.2 A, c = 171.8 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one molecule of enzyme per asymmetric unit. The breakdown of a chromogenic cephalosporin derivative diffused into a crystal reveals clearly that the enzyme is active in the crystalline state. [less ▲]

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See detailCloning and overexpression of the triosephosphate isomerase genes from psychrophilic and thermophilic bacteria. Structural comparison of the predicted protein sequences
Rentier-Delrue, Françoise ULg; Mande, Shekhar C; Moyens, Sylvianne et al

in Journal of Molecular Biology (1993), 229(1), 85-93

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments ... [more ▼]

We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequence determined. Its deduced amino acid sequence revealed 34% identity with the thermophilic bacteria Bacillus stearothermophilus TIM. Expression vectors were constructed and recombinant Moraxella TA137 and Bacillus stearothermophilus TIMs were overproduced and purified to homogeneity. Recombinant TIM inactivation constants (Ki), measured at various temperatures, compared to those of the mesophilic Escherichia coli recombinant TIM clearly show that Moraxella TA137 and B. stearothermophilus TIMs have respectively psychrophilic and thermophilic characteristics. To try to elucidate the structure-thermolability and structure-thermostability relationship, factors affecting the overall stability of these two TIMs were examined, based on the alignment with the mesophilic chicken TIM, the three-dimensional structure of which is already known. From this comparison, it appears that the adaptability of TIM to high temperature is favored by better stabilizing residues for the helix dipole as well as better helix-forming residues whereas the adaptability of TIM to low temperature seems to reside in the nature of helix-capping residues. [less ▲]

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See detailMolecular size and symmetry of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase. An X-ray crystallography analysis.
Marcq, S.; Diaz-Ruano, A.; Charlier, Paulette ULg et al

in Journal of Molecular Biology (1991), 220(1), 9-12

The catabolic ornithine carbamoyltransferase (EC 2.1.3.3) from Pseudomonas aeruginosa, that shows allosteric behaviour, and a mutant version of this enzyme has been crystallized in several different ... [more ▼]

The catabolic ornithine carbamoyltransferase (EC 2.1.3.3) from Pseudomonas aeruginosa, that shows allosteric behaviour, and a mutant version of this enzyme has been crystallized in several different crystal forms. All of these have been characterized by X-ray diffraction methods. A 4.5 A resolution data set has been collected on a triclinic crystal. Analysis of the data using the self-rotation function shows that 12 monomers associate to form a particle with cubic 23 point group symmetry. [less ▲]

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See detailCo-operative binding of the glucocorticoid receptor DNA binding domain is one of at least two mechanisms for synergism.
Baniahmad, Claudia; Muller, Marc ULg; Altschmied, Joachim et al

in Journal of Molecular Biology (1991), 222(2), 155-65

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor binding sequences with adjacent binding sites either for other transcription factors or for further ... [more ▼]

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor binding sequences with adjacent binding sites either for other transcription factors or for further steroid receptors. Analysis of the human glucocorticoid receptor revealed that the DNA-binding domain of the receptor is sufficient to mediate co-operative binding to adjacent receptor binding sites. This is a novel feature of the domain in addition to its DNA-binding, trans-activating and trans-repressing properties. Chimaeric proteins containing the N- or C-terminal receptor halves fused to the GAL4 DNA-binding domain do not co-operate in DNA-binding, however they do functionally synergize. Thus, at least two mechanisms contribute to the synergism of the human glucocorticoid receptor bound to two adjacent receptor binding sites. [less ▲]

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See detailCrystallographic mapping of beta-lactams bound to a d-alanyl-d-alanine peptidase target enzyme
Kelly, Judith A.; Knox, James R.; Zhao, Haiching C. et al

in Journal of Molecular Biology (1989), 209(2), 281-295

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins ... [more ▼]

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins. Cephalosporin C, the monobactam analog of penicillin G and (2,3)-alpha-methylene benzylpenicillin have been mapped at 2.3 A resolution in the form of acyl-enzyme complexes bound to serine 62. On the basis of the positions of these inhibitors, the binding of a tripeptide substrate for the enzyme, L-lysyl-D-alanyl-D-alanine, has been modeled in the active site. The binding of both inhibitors and substrate is facilitated by hydrogen-bonding interactions with a conserved beta-strand (297-303), which is antiparallel to the beta-lactam's acylamide linkage or the substrate's peptide bond. The active site is similar to that in beta-lactamases. [less ▲]

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See detailCrystallization and preliminary X-ray data for the exocellular beta-lactamase of Bacillus licheniformis 749/C.
Dideberg, O.; Libert, M.; Frère, Jean-Marie ULg et al

in Journal of Molecular Biology (1985), 181(1), 145-6

The exocellular beta-lactamase from Bacillus licheniformis 749/C has been crystallized from polyethylene glycol solution at pH 5.5. An X-ray examination of the monoclinic crystals shows the space group is ... [more ▼]

The exocellular beta-lactamase from Bacillus licheniformis 749/C has been crystallized from polyethylene glycol solution at pH 5.5. An X-ray examination of the monoclinic crystals shows the space group is P21, with unit cell dimensions a = 66.77 A, b = 93.77 A, c = 43.57 A and beta = 104.5 degrees. The asymmetric unit consists of two molecules of 28,500 Mr each. The crystals are suitable for structure analysis to at least 2 A resolution. [less ▲]

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See detailCrystallographic data for the beta-lactamase from Enterobacter cloacae P99.
Charlier, Paulette ULg; Dideberg, O.; Frère, Jean-Marie ULg et al

in Journal of Molecular Biology (1983), 171(2), 237-8

The beta-lactamase from Enterobacter cloacae P99 has been crystallized from polyethylene glycol solution at pH 7. X-ray examination of the orthorhombic crystals shows the space group is P2(1)2(1)2 with ... [more ▼]

The beta-lactamase from Enterobacter cloacae P99 has been crystallized from polyethylene glycol solution at pH 7. X-ray examination of the orthorhombic crystals shows the space group is P2(1)2(1)2 with unit cell dimensions a = 77.4 A, b = 69.4 A, and c = 63.6 A. There is one molecule of molecular weight 39,000 in the asymmetric unit. [less ▲]

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See detailCrystallization and preliminary X-ray data for parvalbumin IIIf of Opsanus tau.
Hamoir, G.; Dideberg, O.; Charlier, Paulette ULg

in Journal of Molecular Biology (1981), 153(2), 487-9

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See detailCrystallographic data for the DD-carboxypeptidase-endopeptidase of low penicillin sensitivity excreted by Streptomyces albus g
Dideberg, Otto; Frère, Jean-Marie ULg; Ghuysen, Jean-Marie ULg

in Journal of Molecular Biology (1979), 129(4), 677-679

The DD-carboxypeptidase-endopeptidase of low penicillin sensitivity that is excreted by Streptomyces albus G has been crystallized from a polyethylene glycol (Mr 6000 to 7500) solution at pH 8.0. X-ray ... [more ▼]

The DD-carboxypeptidase-endopeptidase of low penicillin sensitivity that is excreted by Streptomyces albus G has been crystallized from a polyethylene glycol (Mr 6000 to 7500) solution at pH 8.0. X-ray examination of the prismatic crystals shows that the space group is P21 with unit cell dimensions a = 51.1 Å, B = 49.7 Å, C = 38.7 Å, β = 100.6 ° and one molecule in the asymmetric unit. A crystal suspension made in 50 mM -Tris • HCl buffer (pH 8.0) supplemented with 5 mM-MgCl2 and 16% (w/v) polyethylene glycol exhibits enzyme activity on the substrate Ac2-L-Lys-D-Ala-D-Ala. [less ▲]

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See detailCrystallographic data for a penicillin receptor : exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61
Knox, James R.; DeLucia, Mary L.; Murthy, N. S. et al

in Journal of Molecular Biology (1979), 127(2), 217-218

A pencillin-sensitive enzyme, the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7•6. X-ray ... [more ▼]

A pencillin-sensitive enzyme, the exocellular DD-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7•6. X-ray examination of the orthorhombic crystals shows the space group is P212121, with unit cell dimensions a = 51•1 Å, B = 67•4 Å, and C = 102•9 Å. With four molecules of molecular weight 38,000, the Å3/dalton ratio for the cell is 2•33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2•4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å. [less ▲]

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