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See detailTem-1 Beta-Lactamase Folds in a Nonhierarchical Manner with Transient Non-Native Interactions Involving the C-Terminal Region
Lejeune, Annabelle ULg; Pain, R. H.; Charlier, Paulette ULg et al

in Biochemistry (2008), 47(4), 1186-93

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and ... [more ▼]

The conformational stability and kinetics of refolding and unfolding of the W290F mutant of TEM-1 beta-lactamase have been determined as a function of guanidinium chloride concentration. The activity and spectroscopic properties of the mutant enzyme did not differ significantly from those of the wild type, indicating that the mutation has only a very limited effect on the structure of the protein. The stability of the folded protein is reduced, however, by 5-10 kJ mol-1 relative to that of the molten globule intermediate (H), but the values of the folding rate constants are unchanged, suggesting that Trp-290 becomes organized in its nativelike environment only after the rate-limiting step; i.e., the C-terminal region of the enzyme folds very late. In contrast to the significant increase in fluorescence intensity seen in the dead time (3-4 ms) of refolding of the wild-type protein, no corresponding burst phase was observed with the mutant enzyme, enabling the burst phase to be attributed specifically to the C-terminal Trp-290. This residue is suggested to be buried in a nonpolar environment from which it has to escape during subsequent folding steps. With both proteins, fast early collapse leads to a folding intermediate in which the C-terminal region of the polypeptide chain is trapped in a non-native structure, consistent with a nonhierarchical folding process. [less ▲]

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See detailEngineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils
Chan, Pak Ho; Pardon, Els; Menzer, Linda ULg et al

in Biochemistry (2008), 47

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic ... [more ▼]

single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation. [less ▲]

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See detailActivation mechanism of recombinant Der p 3 allergen zymogen - Contribution of cysteine protease Der p 1 and effect of propeptide glycosylation
Dumez, Marie-Eve ULg; Teller, Nathalie; Mercier, Frédéric ULg et al

in Journal of Biological Chemistry (2008), 283(45), 30606-30617

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been ... [more ▼]

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence TP1R. [less ▲]

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See detailRelationship between propeptide pH unfolding and inhibitory ability during ProDer p 1 activation mechanism
Chevigné, Andy ULg; Barumandzadeh, Roya ULg; Groslambert, Sylvie et al

in Journal of Molecular Biology (2007), 374(1), 170-185

The major allergen Der p1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The ... [more ▼]

The major allergen Der p1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p I exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propepticles with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K-D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propepticle characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding. (c) 2007 Elsevier Ltd. All rights reserved. [less ▲]

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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 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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