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See detailDetermination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae.
de Ruyck, Jerome; Janczak, Matthew W.; Neti, Syam Sundar et al

in Chembiochem : a European journal of chemical biology (2014), 15(10), 1452-1458

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1 ... [more ▼]

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI-2) is a flavoenzyme found in bacteria that is completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady-state kinetic studies of the enzyme indicated that FMNH2 (KM =0.3 muM) bound before isopentenyl diphosphate (KM =40 muM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 A resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence-similar and structure-related pathogens such as Enterococcus faecalis or Staphylococcus aureus. [less ▲]

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See detailClass A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine
Huynen, Céline ULg; Filée, Patrice; Matagne, André ULg et al

in BioMed Research International (2013), 2013

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽 ... [more ▼]

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽-lactamases as versatile scaffolds to design hybrid enzymes (referred to as 𝛽-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions.We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins. [less ▲]

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See detailA Nanobody Binding to Non-amyloidogenic Regions of the Protein Human Lysozyme Enhances Partial Unfolding but Inhibits Amyloid Fibril Formation.
de Genst, EJ; Chan, PH; Pardon, Els et al

in Journal of Physical Chemistry B (2013)

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See detailThe proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.
Dumez, Marie-Eve ULg; Herman, Julie; Campisi, Vincenzo ULg et al

in PloS one (2013), 8(9), 68014

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific ... [more ▼]

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen. [less ▲]

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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 detailEnergetics of protein stability at extreme environmental temperatures in bacterial trigger factors
Struvay, Caroline ULg; Negro, Sonia; Matagne, André ULg et al

in Biochemistry (2013), 52

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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 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 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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