References of "Matagne, André"
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See detailTowards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case.
Roussel, Guillaume; Perpete, Eric A.; Matagne, André ULg et al

in Biotechnology and Bioengineering (2013), 110(2), 417-23

It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha ... [more ▼]

It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD-based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far-UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20-40 degrees C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417-423. (c) 2012 Wiley Periodicals, Inc. [less ▲]

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See detailIn silico and in vivo combinatorial design of Octarellin VI, an artificial protein modeled on the (B/A)8 fold
Figueroa Yévenes, Maximiliano ULg; Taralla, Sébastien; Buscetta, Marco et al

Poster (2012, November 16)

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in ... [more ▼]

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design. [less ▲]

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See detailFolding and Stability of Class A beta-Lactamases
Matagne, André ULg; Pain, Roger

in Frère, Jean-Marie (Ed.) Beta-Lactamases (2012)

Class A β-lactamases have proved useful as model proteins in studying a wide variety of aspects of protein folding. We review those features that have shed light on kinetic intermediates that take part in ... [more ▼]

Class A β-lactamases have proved useful as model proteins in studying a wide variety of aspects of protein folding. We review those features that have shed light on kinetic intermediates that take part in folding, including some insight into the molecular basis of the kinetic and stable molten-globule states that have been identified. The contrast between the folding behaviour of PC1 and the two lactamase mutants, P54 and P2, can be attributed in some detail to changes in molecular conformation. The early, very rapid stages of folding of β-lactamases have been shown to be multiphasic, and an interesting intermediate is described that has non-native contacts involving burial of the C-terminal tryptophan. A further feature is that the region around the disulphide bond in the TEM enzymes is formed very early in the foldingreaction. The unusual feature of class A β-lactamases, i.e. a cispeptide bond critical in the stereochemistry of the active site that usually, but not always, involves a proline residue, has been shown to be important in accounting for the slow folding reactions. The effect of substrates on the stabilization of the enzymes and on their reversible deactivation is also reviewed. [less ▲]

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See detailPurification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.
Roussel, G; Matagne, André ULg; De Bolle, X et al

in Protein Expression & Purification (2012), 83(2), 198-204

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their ... [more ▼]

Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella beta-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-beta-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination. [less ▲]

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See detailStructural characterization of recombinant bovine Goalpha by spectroscopy and homology modeling
Tiber, Pinar Mega; Orun, Oya; Nacar, Cevdet et al

in Spectroscopy (2011), 26

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See detailEffects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.
Vandevenne, Marylène ULg; GASPARD, Genevieve ULg; Belgsir, E. M. et al

in Biochimica et biophysica acta (2011)

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations ... [more ▼]

Irreversible accumulation of protein aggregates represents an important problem both in vivo and in vitro. The aggregation of proteins is of critical importance in a wide variety of biomedical situations, ranging from diseases (such as Alzheimer's and Parkinson's diseases) to the production (e.g. inclusion bodies), stability, storage and delivery of protein drugs. beta-Cyclodextrin (beta-CD) is a circular heptasaccharide characterized by a hydrophilic exterior and a hydrophobic interior ring structure. In this research, we studied the effects of a chemically modified beta-CD (BCD07056), on the aggregating and refolding properties of BlaPChBD, a hybrid protein obtained by inserting the chitin binding domain of the human macrophage chitotriosidase into the class A beta-lactamase BlaP from Bacillus licheniformis 749/I during its thermal denaturation. The results show that BCD07056 strongly increases the refolding yield of BlaPChBD after thermal denaturation and constitutes an excellent additive to stabilize the protein over time at room temperature. Our data suggest that BCD07056 acts early in the denaturation process by preventing the formation of an intermediate which leads to an aggregated state. Finally, the role of beta-CD derivatives on the stability of proteins is discussed. [less ▲]

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See detailStepwise adaptations to low temperature as revealed by multiple mutants of a psychrophilic alpha-amylase from an Antarctic bacterium
Cipolla, Alexandre ULg; D'Amico, Salvino ULg; Barumandzadeh, Roya et al

in Journal of Biological Chemistry (2011), 286(44), 3834838355

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See detailSelective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex.
Azarkan, Mohamed; Matagne, André ULg; Wattiez, Ruddy et al

in Phytochemistry (2011), 72(14-15), 1718-31

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family ... [more ▼]

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors. [less ▲]

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See detailThree factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys 70
Vercheval, Lionel ULg; Di Paolo, Alexandre ULg; Borel, Franck et al

in Biochemical Journal (2010), 432(3), 495-504

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by ... [more ▼]

Lys-70 carboxylation in the active site of class D β lactamases is essential for their activity. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val-117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys 70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate limiting step for the wild type OXA 10 β lactamase. [less ▲]

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See detailGeneration of camelid single-domain antibody fragments raised against proteins containing polyglutamine expansions
Pain, Coralie ULg; Scarafone, Natacha; Jaspar, Aurélie et al

Poster (2010, October 14)

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins ... [more ▼]

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins with expanded polyQ repeats have been found to form intranuclear amyloid-like aggregates, and the formation of these aggregates could play an important role in the pathogenesis [2-4]. The polyQ expansion is the only common feature among the proteins involved, suggesting it may be responsible for the aggregation phenomenon. Understanding the molecular mechanism by which the polyQ expansions promote aggregation is therefore crucial for the development of therapeutic strategies. The nine proteins associated with polyQ diseases are difficult to express recombinantly due to their big size and/or their insoluble character. In order to get further insights into the mechanism by which polyQ tracts promote aggregation, we have therefore decided to insert polyQ sequences into a well studied protein, the b-lactamase BlaP from B. licheniformis [5-6]. We have created chimeras containing 23, 30, 55, and 79 glutamines and we have investigated the effects of the insertions on the activity, the structure, the stability of BlaP as well as on its aggregating properties. Preliminary results indicate that BlaP is a good framework to study the molecular mechanism of aggregation associated with expanded polyglutamine tracts. On another hand, our previous work on the amyloidogenic variants of human lysozyme has shown that camelid single domain antibody fragments are very powerful structural probes to understand, at the molecular level, the mechanism of amyloid fibril formation [7]. Moreover, a recent study has suggested that expanded polyQ strectches adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies [8]. Altogether these results have encouraged us to generate VHHs against our different chimeras and we present here our preliminary results. References [1] Orr and Zoghbi (2007) Annu Rev Neurosci 30, 575-621. [2] DiFiglia et al. (1997) Science 277, 1990-1993. [3] Paulson HL (2000) Brain Pathol 10, 293-299. [4] Sanchez I. et al. (2003) Nature 421, 373-379. [5] Scarafone N. (2008) Mémoire de DEA en Sciences. Université de Liège. [6] Pain C. (2009) Mémoire de Master en Biochimie. Université de Liège. [7] Dumoulin et al. (2003) Nature 424, 783-788. [8] Legleiter J. et al. (2009) J Biol Chem 284, 21647-21648. [less ▲]

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See detailConsistent picture of the reversible thermal unfolding of hen egg-white lysozyme from experiment and molecular dynamics
Meersman, Filip; Atilgan, Canan; Miles, Andrew J. et al

in Biophysical Journal (2010), 99

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See detailBackbone 1H, 13C, and 15N resonance assignments for lysozyme from bacteriophage lambda.
Di Paolo, Alexandre ULg; Duval, Valerie; Matagne, André ULg et al

in Biomolecular NMR assignments (2010), 4(1), 111-4

Lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two ... [more ▼]

Lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of lambda lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes lambda lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of lambda lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the (1)H, (13)C and (15)N backbone resonance assignments for lambda lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR. [less ▲]

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See detail1H, 13C and 15N backbone resonance assignments for the BS3 class A beta-lactamase from Bacillus licheniformis.
Vandenameele, Julie ULg; Matagne, André ULg; Damblon, Christian ULg

in Biomolecular NMR Assignments (2010), 4(2), 195-7

Class A beta-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-alpha domain ... [more ▼]

Class A beta-lactamases (260-280 amino acids; M ( r ) ~ 29,000) are among the largest proteins studied in term of their folding properties. They are composed of two structural domains: an all-alpha domain formed by five to eight helices and an alpha/beta domain consisting of a five-stranded antiparallel beta-sheet covered by three to four alpha-helices. The alpha domain (~150 residues) is made up of the central part of the polypeptide chain whereas the alpha/beta domain (111-135 residues) is constituted by the N- and C-termini of the protein. Our goal is to determine in which order the different secondary structure elements are formed during the folding of BS3. With this aim, we will use pulse-labelling hydrogen/deuterium exchange experiments, in combination with 2D-NMR measurements, to monitor the time-course of formation and stabilization of secondary structure elements. Here we report the backbone resonance assignments as the requirement for further hydrogen/deuterium exchange studies. [less ▲]

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See detailRapid Collapse into a Molten Globule Is Followed by Simple Two-State Kinetics in the Folding of Lysozyme from Bacteriophage lambda
Di Paolo, Alexandre ULg; Balbeur, D.; De Pauw, Edwin ULg et al

in Biochemistry (2010), 49

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass ... [more ▼]

Stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional NMR and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage lambda (lambda lysozyme) at pH 5.6, 20 degrees C. The first step in the folding of lambda lysozyme occurs very rapidly (tau < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of secondary structure but only weak protection from (1)H/(2)H exchange and no fixed tertiary structure organization. This early folding step is reflected in the dead-time events observed in the far-UV CD and ANS fluorescence experiments. Following accumulation of this kinetic molten globule species, the secondary structural elements are stabilized and the majority (ca. 88%) of refolding molecules acquire native-like properties in a highly cooperative two-state process, with tau = 0.15 +/- 0.03 s. This is accompanied by the acquisition of substantial native-like protection from hydrogen exchange. A double-mixing experiment and the absence of a denaturant effect reveal that slow (tau = 5 +/- 1 s) folding of the remaining (ca. 12%) molecules is rate limited by the cis/trans isomerization of prolines that are trans in the folded enzyme. In addition, native state hydrogen exchange and classical denaturant unfolding experiments have been used to characterize the thermodynamic properties of the enzyme. In good agreement with previous crystallographic evidence, our results show that lambda lysozyme is a highly dynamic protein, with relatively low conformational stability (DeltaG degrees (N-U) = 25 +/- 2 kJ.mol(-1)). [less ▲]

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See detailFolding of class A beta-lactamases is rate-limited by peptide bond isomerization and occurs via parallel pathways.
Vandenameele, Julie ULg; Lejeune, Annabelle ULg; Di Paolo, Alexandre ULg et al

in Biochemistry (2010), 49(19), 4264-75

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues ... [more ▼]

Class A beta-lactamases (M(r) approximately 29000) provide good models for studying the folding mechanism of large monomeric proteins. In particular, the highly conserved cis peptide bond between residues 166 and 167 at the active site of these enzymes controls important steps in their refolding reaction. In this work, we analyzed how conformational folding, reactivation, and cis/trans peptide bond isomerizations are interrelated in the folding kinetics of beta-lactamases that differ in the nature of the cis peptide bond, which involves a Pro167 in the BS3 and TEM-1 enzyme, a Leu167 in the NMCA enzyme, and which is missing in the PER-1 enzyme. The analysis of folding by spectroscopic probes and by the regain of enzymatic activity in combination with double-mixing procedures indicates that conformational folding can proceed when the 166-167 bond is still in the incorrect trans form. The very slow trans --> cis isomerization of the Glu166-Xaa167 peptide bond, however, controls the final step of folding and is required for the regain of the enzymatic activity. This very slow phase is absent in the refolding of PER-1, in which the Glu166-Ala167 peptide bond is trans. The double-mixing experiments revealed that a second slow kinetic phase is caused by the cis/trans isomerization of prolines that are trans in the folded proteins. The folding of beta-lactamases is best described by a model that involves parallel pathways. It highlights the role of peptide bond cis/trans isomerization as a kinetic determinant of folding. [less ▲]

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