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See detailOctarellin VI: using rosetta to design a putative artificial (beta/alpha)8 protein.
Figueroa Yévenes, Maximiliano ULg; Oliveira, Nicolas; Lejeune, Annabelle et al

in PloS one (2013), 8(8), 71858

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting ... [more ▼]

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with alpha-helical and beta-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70 degrees C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial alpha/beta protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility. [less ▲]

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See detailThe dynamics of lysozyme from bacteriophage lambda in solution probed by NMR and MD simulations.
Smith, Lorna J.; Bowen, Alice M.; Di Paolo, Alexandre et al

in Chembiochem : a European journal of chemical biology (2013), 14(14), 1780-8

(15) N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H(N) -H(alpha) coupling constants, and molecular dynamics (MD) simulations have ... [more ▼]

(15) N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H(N) -H(alpha) coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (lambda lysozyme) in solution. The lower and upper lip regions in lambda lysozyme (residues 51-60 and 128-141, respectively) show reduced (1) H-(15) N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T2 indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for lambda lysozyme indicate that two fluctuating beta-strands (beta3 and beta4) are populated in the lower lip region while the N terminus of helix alpha6 (residues 136-139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the beta-sheet and helical secondary structure in the lip regions, with populations of 40-60 %. Thus in solution lambda lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein. [less ▲]

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See detailEfficient refolding of a recombinant abzyme : Structural and catalytic characterizations.
Ben Naya, Raouia; Matti, Kalyankumar; Guellier, Adeline et al

in Applied Microbiology & Biotechnology (2013), 97(17), 7721-31

Catalytic antibodies are currently being investigated in order to understand their role under physio-pathological situations. To this end, the knowledge of structure-function relationships is of great ... [more ▼]

Catalytic antibodies are currently being investigated in order to understand their role under physio-pathological situations. To this end, the knowledge of structure-function relationships is of great interest. Recombinant scFv fragments are smaller and easier to genetically manipulate than whole antibodies, making them well suited for this kind of study. Nevertheless they are often described as proteins being laborious to produce. This paper describes a highly efficient method to produce large quantities of refolded soluble catalytic scFv. For the first time, the functionality of a refolded catalytic scFv displaying a beta-lactamase activity has been validated by three approaches: (1) use of circular dichroism to ensure that the refolded had secondary structure consistent with a native scFv fold, (2) development of enzyme-linked immunosorbant assay and surface plasmon resonance (SPR) approaches for testing that the binding characteristics of an inhibitory peptide have been retained, and (3) proof of the subtle catalytic properties conservation through the development of a new sensitive catalytic assay using a fluorogenic substrate. [less ▲]

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See detailStructural Determinants of Specificity and Catalytic Mechanism in mammalian 25-kDa Thiamine Triphosphatase
Delvaux, David; Kerff, Frédéric ULg; Murty, Mamidanna R.V.S. et al

in Biochimica et Biophysica Acta - General Subjects (2013), 1830

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine ... [more ▼]

Background: Thiamine triphosphate (ThTP) is present in most organisms and might be involved in intracellular signaling. In mammalian cells, the cytosolic ThTP level is controlled by a specific thiamine triphosphatase (ThTPase), belonging to the CYTH superfamily of proteins. CYTH proteins are present in all superkingdoms of life and act on various triphosphorylated substrates. Methods: Using crystallography, mass spectrometry and mutational analysis, we identified the key structural determinants of the high specificity and catalytic efficiency of mammalian ThTPase. Results: Triphosphate binding requires three conserved arginines while the catalytic mechanism relies on an unusual lysine-tyrosine dyad. By docking of the ThTP molecule in the active site, we found that Trp-53 should interact with the thiazole part of the substrate molecule, thus playing a key role in substrate recognition and specificity. Sea anemone and zebrafish CYTH proteins, which retain the corresponding Trp residue, are also specific ThTPases. Surprisingly, the whole chromosome region containing the ThTPase gene is lost in birds. Conclusion: The specificity for ThTP is linked to a stacking interaction between the thiazole heterocycle of thiamine and a tryptophan residue. The latter likely plays a key role in the secondary acquisition of ThTPase activity in early metazoan CYTH enzymes, in the lineage leading from cnidarians to mammals. General significance: We show that ThTPase activity is not restricted to mammals as previously thought but is an acquisition of early metazoans. This, and the identification of critically important residues, allows us to draw an evolutionary perspective of the CYTH family of proteins. [less ▲]

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See detailUse of red autofluorescence for monitoring prodiginine biosynthesis
Tenconi, Elodie ULg; Guichard, Paul; Motte, Patrick ULg et al

in Journal of Microbiological Methods (2013), 93

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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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