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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 detailGuanidinium chloride denaturation of the dimeric Bacillus licheniformis Blal repressor highlights an independent domain unfolding pathway
Vreuls, Christelle ULg; Filée, Patrice ULg; Van Melckebeke, H. et al

in Biochemical Journal (2004), 384(Pt 1), 179-190

The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase ... [more ▼]

The Bacillus licheniformis 74911 BlaI repressor is a prokaryotic regulator that, in the absence of a P-lactam antibiotic, prevents the transcription of the blaP gene, which encodes the BlaP beta-lactamase. The BlaI repressor is composed of two structural domains. The 82-residue NTD (N-terminal domain) is a DNA-binding domain, and the CTD (C-terminal domain) containing the next 46 residues is a dimerization domain. Recent studies have shown the existence of the monomeric, dimeric and tetrameric forms of BlaI in solution. In the present study, we analyse the equilibrium unfolding of BlaI in the presence of GdmCl (guanidinium chloride) using different techniques: intrinsic and ANS (8-anilinonaphthalene-1-sulphonic acid) fluorescence, far- and near-UV CD spectroscopy, cross-linking, analytical ultracentrifugation, size exclusion chromatography and NMR spectroscopy. In addition, the intact NTD and CTD were purified after proteolysis of BlaI by papain, and their unfolding by GdmCl was also studied. GdmCl-induced equilibrium unfolding was shown to be fully reversible for BlaI and for the two isolated fragments. The results demonstrate that the NTD and CTD of BlaI fold/unfold independently in a four-step process, with no significant cooperative interactions between them. During the first step, the unfolding of the Blal CTD occurs, followed in the second step by the formation of an 'ANS-bound' intermediate state. Crosslinking and analytical ultracentrifugation experiments suggest that the dissociation of the dimer into two partially unfolded monomers takes place in the third step. Finally, the unfolding of the Blal NTD occurs at a GdmCI concentration of approx. 4 M. In summary, it is shown that the Blal CTD is structured, more flexible and less stable than the NTD upon GdmCI denaturation. These results contribute to the characterization of the Blal dimerization domain (i.e. CTD) involved in the induction process. [less ▲]

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See detailSecondary-structure characterization by far-UV CD of highly purified uncoupling protein 1 expressed in yeast
Douette, Pierre ULg; Navet, Rachel ULg; Bouillenne, Fabrice ULg et al

in Biochemical Journal (2004), 380(Pt 1), 139-145

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His(6) epitope at its C-terminus in ... [more ▼]

The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His(6) epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95 %). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCPI-His(6) retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of a-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68 %, which is at least 25 % higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat. [less ▲]

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See detailA camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme
Dumoulin, Mireille ULg; Last, A. M.; Desmyter, A. et al

in Nature (2003), 424(6950), 783-788

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues(1-3), often with serious ... [more ▼]

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues(1-3), often with serious pathological consequences. Non-neuropathic systemic amyloidosis (4-6) is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody(7-9) raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H. Our structural studies reveal that the epitope includes neither the site of mutation nor most residues in the region of the protein structure that is destabilized by the mutation. Instead, the binding of the antibody fragment achieves its effect by restoring the structural cooperativity characteristic of the wild-type protein. This appears to occur at least in part through the transmission of long-range conformational effects to the interface between the two structural domains of the protein. Thus, reducing the ability of an amyloidogenic protein to form partly unfolded species can be an effective method of preventing its aggregation, suggesting approaches to the rational design of therapeutic agents directed against protein deposition diseases. [less ▲]

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See detailSingle-domain antibody fragments with high conformational stability.
Dumoulin, Mireille ULg; Conrath, Katja; Van Meirhaeghe, Annemie et al

in Protein Science : A Publication of the Protein Society (2002), 11(3), 500-15

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been ... [more ▼]

A variety of techniques, including high-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy, have been used to investigate the equilibrium folding properties of six single-domain antigen binders derived from camelid heavy-chain antibodies with specificities for lysozymes, beta-lactamases, and a dye (RR6). Various denaturing conditions (guanidinium chloride, urea, temperature, and pressure) provided complementary and independent methods for characterizing the stability and unfolding properties of the antibody fragments. With all binders, complete recovery of the biological activity after renaturation demonstrates that chemical-induced unfolding is fully reversible. Furthermore, denaturation experiments followed by optical spectroscopic methods and affinity measurements indicate that the antibody fragments are unfolded cooperatively in a single transition. Thus, unfolding/refolding equilibrium proceeds via a simple two-state mechanism (N <--> U), where only the native and the denatured states are significantly populated. Thermally-induced denaturation, however, is not completely reversible, and the partial loss of binding capacity might be due, at least in part, to incorrect refolding of the long loops (CDRs), which are responsible for antigen recognition. Most interestingly, all the fragments are rather resistant to heat-induced denaturation (apparent T(m) = 60-80 degrees C), and display high conformational stabilities (DeltaG(H(2)O) = 30-60 kJ mole(-1)). Such high thermodynamic stability has never been reported for any functional conventional antibody fragment, even when engineered antigen binders are considered. Hence, the reduced size, improved solubility, and higher stability of the camelid heavy-chain antibody fragments are of special interest for biotechnological and medical applications. [less ▲]

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See detailQuantitative Analysis of the Stabilization by Substrate of Staphylococcus Aureus Pc1 Beta-Lactamase
Lejeune, Annabelle ULg; Vanhove, Marc; Lamotte-Brasseur, Josette et al

in Chemistry & Biology (2001), 8(8), 831-42

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme ... [more ▼]

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem. [less ▲]

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See detailKinetic Study of Two Novel Enantiomeric Tricyclic Beta-Lactams Which Efficiently Inactivate Class C Beta-Lactamases
Vilar, M.; Galleni, Moreno ULg; Solmajer, T. et al

in Antimicrobial Agents and Chemotherapy (2001), 45(8), 2215-23

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative beta-lactamases and D-alanyl-D-alanine peptidases (DD ... [more ▼]

A detailed kinetic study of the interaction between two ethylidene derivatives of tricyclic carbapenems, Lek 156 and Lek 157, and representative beta-lactamases and D-alanyl-D-alanine peptidases (DD-peptidases) is presented. Both compounds are very efficient inactivators of the Enterobacter cloacae 908R beta-lactamase, which is usually resistant to inhibition. Preliminary experiments indicate that various extended-spectrum class C beta-lactamases (ACT-1, CMY-1, and MIR-1) are also inactivated. With the E. cloacae 908R enzyme, complete inactivation occurs with a second-order rate constant, k(2)/K', of 2 x 10(4) to 4 x 10(4) M(-1) s(-1), and reactivation is very slow, with a half-life of >1 h. Accordingly, Lek 157 significantly decreases the MIC of ampicillin for E. cloacae P99, a constitutive class C beta-lactamase overproducer. With the other serine beta-lactamases tested, the covalent adducts exhibit a wide range of stabilities, with half-lives ranging from long (>4 h with the TEM-1 class A enzyme), to medium (10 to 20 min with the OXA-10 class D enzyme), to short (0.2 to 0.4 s with the NmcA class A beta-lactamase). By contrast, both carbapenems behave as good substrates of the Bacillus cereus metallo-beta-lactamase (class B). The Streptomyces sp. strain R61 and K15 extracellular DD-peptidases exhibit low levels of sensitivity to both compounds. [less ▲]

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See detailBeta-lactamases, an old but ever renascent problem
Matagne, André ULg; Galleni, Moreno ULg; Laraki, Nezha et al

in van Broekhoven, A (Ed.) Novel Frontiers in the Production of Compounds for Biomedical Use (2001)

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See detailBeta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.
Conrath, K. E.; Lauwereys, M.; Galleni, Moreno ULg et al

in Antimicrobial Agents and Chemotherapy (2001), 45(10), 2807-12

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries ... [more ▼]

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases. [less ▲]

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See detailTechnique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases.
Bouillenne, Fabrice ULg; Matagne, André ULg; Joris, Bernard ULg et al

in Journal of Chromatography. B : Biomedical Sciences and Applications (2000), 737(1-2), 261-5

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ... [more ▼]

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples. [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 detailThe Beta-Lactamase Cycle: A Tale of Selective Pressure and Bacterial Ingenuity
Matagne, André ULg; Dubus, Alain; Galleni, Moreno ULg et al

in Natural Product Reports (1999), 16(1), 1-19

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See detailMechanistic Diversity of Beta-Lactamases
Frère, Jean-Marie ULg; Dubus, Alain; Galleni, Moreno ULg et al

in Biochemical Society Transactions (1999), 27(2), 58-63

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See detailThe Folding Process of Hen Lysozyme: A Perspective from the 'New View'
Matagne, André ULg; Dobson, Christopher M.

in Cellular and Molecular Life Sciences : CMLS (1998), 54(4), 363-71

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has ... [more ▼]

How a conformationally disordered polypeptide chain rapidly and efficiently achieves its well-defined native structure is still a major question in modern structural biology. Although much progress has been made towards rationalizing the principles of protein structure and dynamics, the mechanism of the folding process and the determinants of the final fold are not yet known in any detail. One protein for which folding has been studied in great detail by a combination of diverse techniques is hen lysozyme. In this article we review the present state of our knowledge of the folding process of this enzyme and focus in particular on recent experiments to probe some of its specific features. These results are then discussed in the context of the 'new view' of protein folding based on energy surfaces and landscapes. It is shown that a schematic energy surface for lysozyme folding, which is broadly consistent with our experimental data, begins to provide a unified model for protein folding through which experimental and theoretical ideas can be brought together. [less ▲]

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See detailCatalytic Properties of Class a Beta-Lactamases: Efficiency and Diversity
Matagne, André ULg; Lamotte-Brasseur, Josette; Frère, Jean-Marie ULg

in Biochemical Journal (1998), 330((Pt 2)), 581-98

beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the ... [more ▼]

beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics. [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 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 detailContribution of Mutant Analysis to the Understanding of Enzyme Catalysis: The Case of Class a Beta-Lactamases
Matagne, André ULg; Frère, Jean-Marie ULg

in Biochimica et Biophysica Acta (1995), 1246(2), 109-27

Class A beta-lactamases represent a family of well studied enzymes. They are responsible for many antibiotic resistance phenomena and thus for numerous failures in clinical chemotherapy. Despite the facts ... [more ▼]

Class A beta-lactamases represent a family of well studied enzymes. They are responsible for many antibiotic resistance phenomena and thus for numerous failures in clinical chemotherapy. Despite the facts that five structures are known at high resolution and that detailed analyses of enzymes modified by site-directed mutagenesis have been performed, their exact catalytic mechanism remains controversial. This review attempts to summarize and to discuss the many available data. [less ▲]

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See detailKinetic Study of Interaction between Brl 42715, Beta-Lactamases, and D-Alanyl-D-Alanine Peptidases
Matagne, André ULg; Ledent, Philippe; Monnaie, Didier et al

in Antimicrobial Agents and Chemotherapy (1995), 39(1), 227-31

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4 ... [more ▼]

A detailed kinetic study of the interactions between BRL 42715, a beta-lactamase-inhibiting penem, and various beta-lactamases (EC 3.5.2.6) and D-alanyl-D-alanine peptidases (DD-peptidases, EC 3.4.16.4) is presented. The compound was a very efficient inactivator of all active-site serine beta-lactamases but was hydrolyzed by the class B, Zn(2+)-containing enzymes, with very different kcat values. Inactivation of the Streptomyces sp. strain R61 extracellular DD-peptidase was not observed, and the Actinomadura sp. strain R39 DD-peptidase exhibited a low level of sensitivity to the compound. [less ▲]

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See detailSTRUCTURAL AND KINETIC CHARACTERIZATION OF A BETA-LACTAMASE-INHIBITOR PROTEIN
STRYNADKA, N. C. J.; JENSEN, S. E.; JOHNS, K. et al

in Nature (1994), 368(6472), 657-660

THE past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria1, which is due mainly to plasmid- or chromosomally encoded beta-lactamases ... [more ▼]

THE past decade has seen an alarming worldwide increase in resistance to beta-lactam antibiotics among many pathogenic bacteria1, which is due mainly to plasmid- or chromosomally encoded beta-lactamases that specifically cleave penicillin and cephalosporins, rendering them inactive. There is therefore a need to develop new strategies in the design of effective inhibitors of beta-lactamase. All the small-molecule inhibitors in clinical use are not very effective and are rapidly degraded2,3. Furthermore, newly characterized mutants of the plasmid-mediated beta-lactamase TEM-1 are highly resistant to these small-molecule inhibitors, including clavulanic acid and tazobactam4. It has been shown that Streptomyces clavuligerus produces an exocellular beta-lactamase inhibitory protein (BLIP; M(r) 17.5 K)5. Here we present data defining BLIP as the most effective known inhibitor of a variety of beta-lactamases, with K(i) values in the subnanomolar to picomolar range. To identify those features in BLIP that make it such a potent inhibitor, we have determined its molecular structure at 2.1 angstrom resolution. BLIP is a relatively flat molecule with a unique fold, comprising a tandem repeat of a 76-amino-acid domain. Each domain consists of a helix-loop-helix motif that packs against a four-stranded antiparallel beta-sheet (Fig. 1a). To our knowledge, BLIP is the first example of a protein inhibitor having two similarly folded domains that interact with and inhibit a single target enzyme. [less ▲]

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