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See detailReduced global copperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations
Dumoulin, Mireille ULg; Canet, Denis; Last, Alexander M. et al

in Journal of Molecular Biology (2005), 346(3), 773-788

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data ... [more ▼]

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining a-helices that form the core of the a-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, an associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailA metallo-beta-lactamase enzyme in action: Crystal structures of the monozinc carbapenemase CphA and its complex with biapenem
Garau, Gianpiero; Bebrone, Carine ULg; Anne, Christine et al

in Journal of Molecular Biology (2005), 345(4), 785-795

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important ... [more ▼]

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailRationalising Lysozyme Amyloidosis: Insights from the Structure and Solution Dynamics of T70N Lysozyme
Johnson, Russell J.K.; Christodoulou, John; Dumoulin, Mireille ULg et al

in Journal of Molecular Biology (2005), 352

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with ... [more ▼]

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45–51 and 68–75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5 Å. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70–74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the β-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the α-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the β-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo. [less ▲]

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See detailIdentification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies
Saerens, Dirk; Pellis, Mireille; Loris, Remy et al

in Journal of Molecular Biology (2005), 352

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they ... [more ▼]

Camel single-domain antibody fragments (VHHs) are promising tools in numerous biotechnological and medical applications. However, some conditions under which antibodies are used are so demanding that they can be met by only the most robust VHHs. A universal framework offering the required properties for use in various applications (e.g. as intrabody, as probe in biosensors or on micro-arrays) is highly valuable and might be further implemented when employment of VHHs in human therapy is envisaged. We identified the VHH framework of cAbBCII10 as a potential candidate, useful for the exchange of antigen specificities by complementarity determining region (CDR) grafting. Due to the large number of CDRH loop structures present on VHHs, this grafting technique was expected to be rather unpredictable. Nonetheless, the plasticity of the cAbBCII10 framework allows successful transfer of antigen specificity from donor VHHs onto its scaffold. The cAbBCII10 was chosen essentially for its high level of stability (47 kJ/mol), good expression level (5 mg/l in E. coli) and its ability to be functional in the absence of the conserved disulfide bond. All five chimeras generated by grafting CDR-Hs, from donor VHHs belonging to subfamily 2 that encompass 75% of all antigen-specific VHHs, on the framework of cAbBCII10 were functional and generally had an increased thermodynamic stability. The grafting of CDR-H loops from VHHs belonging to other subfamilies resulted in chimeras of reduced antigen-binding capacity. [less ▲]

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See detailSolution structure of human prolactin
Teilum, K.; Hoch, J. C.; Goffin, Vincent et al

in Journal of Molecular Biology (2005), 351(4), 810-23

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance ... [more ▼]

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction. [less ▲]

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See detailCrystal structure of the catalytic domain of MMP-16/MT3-MMP: Characterization of MT-MMP specific features
Lang, R.; Braun, M.; Sounni, Nor Eddine ULg et al

in Journal of Molecular Biology (2004), 336(1), 213-225

Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this ... [more ▼]

Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this indirect effect on the cellular environment, these MT-MMPs degrade extracellular matrix proteins, and their overproduction is associated with tumor growth. We have solved the structure of the catalytic domain (cd) of MT3-MMP/MMP-16 in complex with the hydroxamic acid inhibitor batimastat. CdMT3-MMP exhibits a classical MMP-fold with similarity to MT1-MMP. Nevertheless, it also shows unique properties such as a modified MT-specific loop and a closed S1' specificity pocket, which might help to design specific inhibitors. Some MT-MMP-specific features, derived from the crystal structures of MT-1-MMP determined previously and MT3-MMP, and revealed in recent mutagenesis experiments, explain the impaired interaction of the MT-MMPs with TIMP-1. Docking experiments with proMMP-2 show some exposed loops including the MT-loop of cdMT3-MMP involved in the interaction with the proMMP-2 prodomain in the activation encounter complex. This model might help to understand the experimentally proven importance of the MT-loop for the activation of proMMP-2. (C) 2003 Elsevier Ltd. All rights reserved. [less ▲]

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See detailCrystal structure of a dimeric oxidized form of human peroxiredoxin 5
Evrard, Christine ULg; Capron, Arnaud; Marchand, Cécile et al

in Journal of Molecular Biology (2004), 337

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified ... [more ▼]

Peroxiredoxin 5 is the last discovered mammalian member of an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Mammalian peroxiredoxin 5 has been recently classified as an atypical 2-Cys peroxiredoxin due to the presence of a conserved peroxidatic N-terminal cysteine (Cys47) and an unconserved resolving C-terminal cysteine residue (Cys151) forming an intramolecular disulfide intermediate in the oxidized enzyme. We have recently reported the crystal structure of human peroxiredoxin 5 in its reduced form. Here, a new crystal form of human peroxiredoxin 5 is described at 2.0 Ǻ resolution. The asymmetric unit contains three polypeptide chains. Surprisingly, beside two reduced chains, the third one is oxidized although the enzyme was crystallized under initial reducing conditions in presence of 1 mM 1,4-dithio-DL-threitol. The oxidized polypeptide chain forms an homodimer with a symmetry related one through intermolecular disulfide bonds between Cys47 and Cys151. The formation of these disulfide bonds is accompanied by the partial unwinding of the N-terminal parts of the a2 helix, which in the reduced form, contains the peroxidatic Cys47 and the α6 helix, which is sequentially close to the resolving residue Cys151. In each monomer of the oxidized chain, the C-terminal part including the α6 helix is completely reorganized and is isolated from the rest of the protein on an extended arm. In the oxidized dimer, the arm belonging to the first monomer now appears at the surface of the second subunit and vice versa. [less ▲]

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See detailSolution structural study of BlaI: Implications for the repression of genes involved in beta-lactam antibiotic resistance
Van Melckebeke, H.; Vreuls, Christelle ULg; Gans, P. et al

in Journal of Molecular Biology (2003), 333(4), 711-720

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See detailTemperature adaptation of proteins: Engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase
D'Amico, Salvino ULg; Gerday, Charles ULg; Feller, Georges ULg

in Journal of Molecular Biology (2003), 332(5), 981-988

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic (alpha-amylase) with or without an extra ... [more ▼]

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic (alpha-amylase) with or without an extra disulfide bond specific to warm-blooded animals. Both multiple mutants display large modifications of stability and activity arising from synergic effects in comparison with single mutations. Newly introduced weak interactions and the disulfide bond confer mesophilic-like stability parameters, as shown by increases in the melting point t(m), in the calorimetric enthalpy DeltaH(cal) and in protection against heat inactivation, as well as by decreases in cooperativity and reversibility of unfolding. In addition, both kinetic and thermodynamic activation parameters of the catalyzed reaction are shifted close to the values of the porcine enzyme. This study confirms the central role of weak interactions in regulating the balance between stability and activity of an enzyme in order to adapt to the environmental temperature. (C) 2003 Elsevier Ltd. All rights reserved. [less ▲]

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See detailActivity, stability and flexibility in Glycosidases adapted to extreme thermal environments
Collins, T.; Meuwis, Marie-Alice ULg; Gerday, Charles ULg et al

in Journal of Molecular Biology (2003), 328(2), 419-428

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational ... [more ▼]

To elucidate the strategy of low temperature adaptation for a cold-adapted family 8 xylanase, the thermal and chemical stabilities, thermal inactivation, thermodependence of activity and conformational flexibility, as well as the thermodynamic basis of these processes, were compared with those of a thermophilic homolog. Differential scanning calorimetry, fluorescence monitoring of guanidine hydrochloride unfolding and fluorescence quenching were used, among other techniques, to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a poor stability and a high flexibility. In contrast, the thermophilic enzyme is shown to have a reduced low temperature activity, high stability and a reduced flexibility. These findings agree with the hypothesis that cold-adapted enzymes overcome the quandary imposed by low temperature environments via a global or local increase in the flexibility of their molecular edifice, with this in turn leading to a reduced stability. Analysis of the guanidine hydrochloride unfolding, as well as the thermodynamic parameters of irreversible thermal unfolding and thermal inactivation shows that the driving force for this denaturation and inactivation is a large entropy change while a low enthalpy change is implicated in the low temperature activity. A reduced number of salt-bridges are believed to be responsible for both these effects. Guanidine hydrochloride unfolding studies also indicate that both family 8 enzymes unfold via an intermediate prone to aggregation. (C) 2003 Elsevier Science Ltd. All rights reserved. [less ▲]

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See detailDe novo backbone and sequence design of an idealized alpha/beta-barrel protein: Evidence of stable tertiary structure
Offredi, Fabrice; Dubail, Fabien; Kischel, Philippe ULg et al

in Journal of Molecular Biology (2003), 325(1), 163-174

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized ... [more ▼]

We have designed, synthesized, and characterized a 216 amino acid residue sequence encoding a putative idealized alpha/beta-barrel protein. The design was elaborated in two steps. First, the idealized backbone was defined with geometric parameters representing our target fold: a central eight parallel-stranded beta-sheet surrounded by eight parallel alpha-helices, connected together with short structural turns on both sides of the barrel. An automated sequence selection algorithm, based on the dead-end elimination theorem, was used to find the optimal amino acid sequence fitting the target structure. A synthetic gene coding for the designed sequence was constructed and the recombinant artificial protein was expressed in bacteria, purified and characterized. Far-UV CD spectra with prominent bands at 222nm and 208nm revealed the presence of alpha-helix secondary structures (50%) in fairly good agreement with the model. A pronounced absorption band in the near-UV CD region, arising from immobilized aromatic side-chains, showed that the artificial protein is folded in solution. Chemical unfolding monitored by tryptophan fluorescence revealed a conformational stability (DeltaG(H2O)) of 35kJ/mol. Thermal unfolding monitored by near-UV CD revealed a cooperative transition with an apparent T(m) of 65 degrees C. Moreover, the artificial protein did not exhibit any affinity for the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS), providing additional evidence that the artificial barrel is not in the molten globule state, contrary to previously designed artificial alpha/beta-barrels. Finally, 1H NMR spectra of the folded and unfolded proteins provided evidence for specific interactions in the folded protein. Taken together, the results indicate that the de novo designed alpha/beta-barrel protein adopts a stable three-dimensional structure in solution. These encouraging results show that de novo design of an idealized protein structure of more than 200 amino acid residues is now possible, from construction of a particular backbone conformation to determination of an amino acid sequence with an automated sequence selection algorithm. [less ▲]

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See detailNMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains.
Liepinsh, Edvards; Genereux, Catherine ULg; Dehareng, Dominique ULg et al

in Journal of Molecular Biology (2003), 327(4), 833-42

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway ... [more ▼]

AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria. Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance. Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G). A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates. The fold is related to that of bacteriophage T7 lysozyme. Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail. The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them. PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity. This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L. [less ▲]

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See detailSolution structural study of BlaI: implications for the repression of genes involved in beta-lactam antibiotic resistance.
Melckebeke, Helene Van; Vreuls, Christelle ULg; Gans, Pierre et al

in Journal of Molecular Biology (2003), 333(4), 711-20

beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate ... [more ▼]

beta-Lactamase and penicillin-binding protein PBP2' mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Two repressors (BlaI and MecI) regulate expression of these inducible proteins. Here, we present the first solution structure of the 82 amino acid residue DNA-binding domain of Bacillus licheniformis BlaI which is very similar in primary sequence to the medically significant Staphyloccocal BlaI and MecI proteins. This structure is composed of a compact core of three alpha-helices and a three-stranded beta-sheet typical of the winged helix protein (WHP) family. The protein/DNA complex was studied by NMR chemical shift comparison between the free and complexed forms of BlaI. Residues involved in DNA interaction were identified and a WHP canonical model of interaction with the operators is proposed. In this model, specific contacts occur between the base-pairs of the TACA motif and conserved amino acid residues of the repressor helix H3. These results help toward understanding the repression and induction mechanism of the genes coding for beta-lactamase and PBP2'. [less ▲]

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See detailImpact of mutations affecting ND mitochondria-encoded Subunits on the activity and assembly of complex I in chlamydomonas. Implication for the structural organization of the enzyme
Cardol, Pierre ULg; Matagne, René-Fernand ULg; Remacle, Claire ULg

in Journal of Molecular Biology (2002), 319(5), 1211-1221

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex 1) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five ... [more ▼]

The mitochondrial rotenone-sensitive NADH:ubiquinone oxidoreductase (complex 1) comprises more than 35 subunits, the majority of which are encoded by the nucleus. In Chlamydomonas reinhardtii, only five components (ND1, ND2, ND4, ND5 and ND6) are coded for by the mitochondrial genome. Here, we characterize two mitochondrial mutants (dum5 and dum17) showing strong reduction or inactivation of complex I activity: dum5 is a IT deletion in the 3' UTR of nd5 whereas dum17 is a IT deletion in the coding sequence of nd6. The impact of these mutations and of mutations affecting nd1, nd4 and nd4/nd5 genes on the assembly of complex I is investigated. After separation of the respiratory complexes by blue native (BN)-PAGE or sucrose gradient centrifugation, we demonstrate that the absence of intact ND1 or ND6 subunit prevents the assembly of the 850 kDa whole complex, whereas the loss of ND4 or ND4/ND5 leads to the formation of a subcomplex of 650 kDa present in reduced amount. The implications of our findings for the possible role of these ND subunits on the activity of complex I and for the structural organization of the membrane arm of the enzyme are discussed. In mitochondria from all the strains analyzed, we moreover detected a 160210 kDa fragment comprising the hydrophilic 49 kDa and 76 kDa subunits of the complex I peripheral arm and showing NADH dehydrogenase activity. (C) 2002 Elsevier Science Ltd. All rights reserved. [less ▲]

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See detailThe Disulphide Mapping, Folding and Characterisation of Recombinant Ber e 1, an Allergenic Protein, and SFA8, Two Sulphur-rich 2 S Plant Albumins
Alcocer, Marcos; Murtagh, G. J.; Bailey, Kevin et al

in Journal of Molecular Biology (2002), 324

We have cloned and expressed genes encoding the allergenic brazil nut 2 S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were ... [more ▼]

We have cloned and expressed genes encoding the allergenic brazil nut 2 S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2 S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphurrich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy. [less ▲]

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See detailModular Structure, Local Flexibility and Cold-Activity of a Novel Chitobiase from a Psychrophilic Antarctic Bacterium
Lonhienne, T.; Zoidakis, J.; Vorgias, C. E. et al

in Journal of Molecular Biology (2001), 310(2), 291-7

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature ... [more ▼]

The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains: a catalytic domain stabilized by Ca(2+), a galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results suggest that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low K(m) and high k(cat) values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb. [less ▲]

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See detailCrystal Structure of Human Peroxiredoxin 5, a Novel Type of Mammalian Peroxiredoxin at 1.5 Ǻ Resolution
Declercq, Jean-Paul; Evrard, Christine ULg; Clippe, André et al

in Journal of Molecular Biology (2001), 311

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules ... [more ▼]

The peroxiredoxins define an emerging family of peroxidases able to reduce hydrogen peroxide and alkyl hydroperoxides with the use of reducing equivalents derived from thiol-containing donor molecules such as thioredoxin, glutathione, trypanothione and AhpF. Peroxiredoxins have been identified in prokaryotes as well as in eukaryotes. Peroxiredoxin 5 (PRDX5) is a novel type of mammalian thioredoxin peroxidase widely expressed in tissues and located cellularly to mitochondria, peroxisomes and cytosol. Functionally, PRDX5 has been implicated in antioxidant protective mechanisms as well as in signal transduction in cells. We report here the 1.5 Ǻ resolution crystal structure of human PRDX5 in its reduced form. The crystal structure reveals that PRDX5 presents a thioredoxin-like domain. Interestingly, the crystal structure shows also that PRDX5 does not form a dimer like other mammalian members of the peroxiredoxin family. In the reduced form of PRDX5, Cys47 and Cys151 are distant of 13.8 Ǻ although these two cysteine residues are thought to be involved in peroxide reductase activity by forming an intramolecular disul®de intermediate in the oxidized enzyme. These data suggest that the enzyme would necessitate a conformational change to form a disulfide bond between catalytic Cys47 and Cys151 upon oxidation according to proposed peroxide reduction mechanisms. Moreover, the presence of a benzoate ion, a hydroxyl radical scavenger, was noted close to the active-site pocket. The possible role of benzoate in the antioxidant activity of PRDX5 is discussed. [less ▲]

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See detailA Fast Method To Predict Protein Interaction Sites From Sequences
Gallet, X.; Charloteaux, Benoît ULg; Thomas, Annick ULg et al

in Journal of Molecular Biology (2000), 302(4), 917-26

A simple method for predicting residues involved in protein interaction sites is proposed. In the absence of any structural report, the procedure identifies linear stretches of sequences as "receptor ... [more ▼]

A simple method for predicting residues involved in protein interaction sites is proposed. In the absence of any structural report, the procedure identifies linear stretches of sequences as "receptor-binding domains" (RBDs) by analysing hydrophobicity distribution. The sequences of two databases of non-homologous interaction sites eliciting various biological activities were tested; 59-80 % were detected as RBDs. A statistical analysis of amino acid frequencies was carried out in known interaction sites and in predicted RBDs. RBDs were predicted from the 80,000 sequences of the Swissprot database. In both cases, arginine is the most frequently occurring residue. The RBD procedure can also detect residues involved in specific interaction sites such as the DNA-binding (95 % detected) and Ca-binding domains (83 % detected). We report two recent analyses; from the prediction of RBDs in sequences to the experimental demonstration of the functional activities. The examples concern a retroviral Gag protein and a penicillin-binding protein. We support that this method is a quick way to predict protein interaction sites from sequences and is helpful for guiding experiments such as site-specific mutageneses, two-hybrid systems or the synthesis of inhibitors. [less ▲]

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See detailThermal unfolding of an intermediate is associated with non-arrhenius kinetics in the folding of hen lysozyme
Matagne, André ULg; Jamin, M.; Chung, E. W. et al

in Journal of Molecular Biology (2000), 297(1), 193-210

A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism ... [more ▼]

A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 degrees C to 50 degrees C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 degrees C, it decreases above 40 degrees C. In addition, the transient intermediate on the major folding pathway at 20 degrees C, in which the alpha-domain is persistently structured in the absence of a stable beta-domain, is thermally unfolded in a sigmoidal transition (T-m approximate to 40 degrees C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast (similar to 25%) and slow (similar to 75%) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly fitted to a sequential three-state model for the slow folding pathway. Together with previous findings, these results indicate that the alpha-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the alpha-domain intermediate (the dominant factor at high temperatures). Destablization of such kinetically competent intermediate species is Likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated. (C) 2000 Academic Press. [less ▲]

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See detailLiquid crystalline ordering of procollagen as a determinant of three-dimensional extracellular matrix architecture.
Martin, Raquel; Farjanel, J.; Eichenberger, D. et al

in Journal of Molecular Biology (2000), 301(1), 11-7

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged ... [more ▼]

The precise molecular mechanisms that determine the three-dimensional architectures of tissues remain largely unknown. Within tissues rich in extracellular matrix, collagen fibrils are frequently arranged in a tissue-specific manner, as in certain liquid crystals. For example, the continuous twist between fibrils in compact bone osteons resembles a cholesteric mesophase, while in tendon, the regular, planar undulation, or "crimp", is akin to a precholesteric mesophase. Such analogies suggest that liquid crystalline organisation plays a role in the determination of tissue form, but it is hard to see how insoluble fibrils could spontaneously and specifically rearrange in this way. Collagen molecules, in dilute acid solution, are known to form nematic, precholesteric and cholesteric phases, but the relevance to physiological assembly mechanisms is unclear. In vivo, fibrillar collagens are synthesised in soluble precursor form, procollagens, with terminal propeptide extensions. Here, we show, by polarized light microscopy of highly concentrated (5-30 mg/ml) viscous drops, that procollagen molecules in physiological buffer conditions can also develop long-range nematic and precholesteric liquid crystalline ordering extending over 100 microm(2) domains, while remaining in true solution. These observations suggest the novel concept that supra-fibrillar tissue architecture is determined by the ability of soluble precursor molecules to form liquid crystalline arrays, prior to fibril assembly. [less ▲]

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