The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.
Figueroa Yévenes, Maximiliano ; ; Vandevenne, Marylène et al
in Journal of structural biology (2016)
Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the ... [more ▼]
Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (alphabetaalpha) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features. [less ▲]Detailed reference viewed: 17 (3 ULg)
The N-terminal region of CHD4 is essential for activity and contains a HMG-box-like-domain that can bind poly(ADP-ribose).
; ; et al
in Journal of Biological Chemistry (2015)Detailed reference viewed: 3 (2 ULg)
Enzymatic functionalization of a nanobody using protein insertion technology
Crasson, Oscar ; ; et al
in Protein Engineering, Design & Selection (2015), 28(10), 451-460
Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a ... [more ▼]
Antibody-based products constitute one of the most attractive biological molecules for diagnostic, medical imagery and therapeutic purposes with very few side effects. Their development has be- come a major priority of biotech and pharmaceutical industries. Recently, a growing number of modified antibody-based products have emerged including fragments, multi-specific and conjugate antibodies. In this study, using protein engineering, we have functionalized the anti-hen egg-white lysozyme (HEWL) camelid VHH antibody fragment (cAb-Lys3), by insertion into a solvent-exposed loop of the Bacillus licheniformis β-lactamase BlaP. We showed that the generated hybrid protein conserved its enzymatic activity while the displayed nanobody retains its ability to inhibit HEWL with a nanomolar affinity range. Then, we successfully implemented the functionalized cAb-Lys3 in enzyme-linked immunosorbent assay, potentiometric biosensor and drug screening assays. The hybrid protein was also expressed on the surface of phage particles and, in this context, was able to interact specifically with HEWL while the β-lactamase activity was used to monitor phage interactions. Finally, using thrombin-cleavage sites surrounding the permissive insertion site in the β-lactamase, we reported an expression system in which the nanobody can be easily separated from its carrier protein. Altogether, our study shows that insertion into the BlaP β-lactamase consti- tutes a suitable technology to functionalize nanobodies and allowsthe creation of versatile tools that can be used in innovative biotechnological assays. [less ▲]Detailed reference viewed: 45 (16 ULg)
The hidden face of the human macrophage chitotriosidase: taking a new look at this enzyme based on the biochemical and phylogenomic analysis of its chitin binding domain
Crasson, Oscar ; ; et al
Poster (2015, August)
Carbohydrates recognition is a critical process involved in numerous aspects of the cell biology such as inflammation, innate immune responses and proliferation. Chitin is an homopolysaccharide composed ... [more ▼]
Carbohydrates recognition is a critical process involved in numerous aspects of the cell biology such as inflammation, innate immune responses and proliferation. Chitin is an homopolysaccharide composed of β-1,4-linked N-acetylglucosamine (GlcNAc) units that is an abundant structural component of various infectious organisms like protozoans, nematodes and fungi. As there is no endogenous chitin produced by mammals, this polymer appeared to be a strategic target for innate immune agents which is why various carbohydrate binding proteins, associated or not with catalytic domains, are synthetized by plants and animals and are known to play a crucial role in innate immunity. The macrophage chitotriosidase (HCHT) is one of the three active chitinases synthetized by humans and has triggered significant attention recently due to its association with various inflammatory disorders. HCHT belongs to the Glycosyl Hydrolase family 18 (GH18) and is known to be involved in innate immunity. Nevertheless, its precise physiological function remains unclear. As numerous GHs, HCHT is a modular protein composed of a catalytic domain (GH18) associated to a Carbohydrate Binding Module (CBM) which is essential to hydrolyse crystalline chitin. If the catalytic domain GH18 is highly common in other GHs from animals, plants, fungi, bacteria, archea and viruses, its CBM (named ChBD) is much less conserved which makes the association between these two domains particularly intriguing. This work aims to demystify HCHT’s physiological function. Firstly, using competitive inhibition assays, we have highlighted the ability of ChBD to interact with chitooligosaccharides (GlcNAc1-2-4-6) which suggests that ChBD can potentially act as a lectin domain. Secondly, to better understand the molecular basis for chitin recognition, we have used homology modelling to build, with high confidence, the 3D structure model of ChBD. Based on this model, a specific set of residues has been selected for alanine scan mutagenesis which has allowed us to define the minimum chitin binding interface of the protein. Thirdly, Phylogenomic studies were performed to analyse the evolutionary history of the isolated catalytic and ChBD domains and understand how these domains were combined. Based on all these results, we discuss a new way of looking at HCHT where its ChBD would be the key determinant that has guided the catalytic domain from a basic metabolic function to a critical component of innate immunity in human. Finally, we propose a mechanism that explains how this enzyme could act at the molecular level to defend us against chitin-containing pathogens. [less ▲]Detailed reference viewed: 98 (21 ULg)
Site directed nitroxide spin labeling of oligonucleotides for NMR and EPR studies
; ; Vandevenne, Marylène et al
in Tetrahedron (2015), 71(5), 813-819Detailed reference viewed: 4 (1 ULg)
How to build a biological linker dedicated to the engineering of novel drug delivery systems
Crasson, Oscar ; Galleni, Moreno ; et al
Poster (2014, May 15)Detailed reference viewed: 99 (20 ULg)
Class A beta-lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine (Invited speaker)
Conference (2014, May 05)Detailed reference viewed: 4 (0 ULg)
Engineering Specificity Changes on a RanBP2 Zinc Finger that Binds Single-Stranded RNA.
Vandevenne, Marylène ; ; et al
in Angewandte Chemie International Edition (2014)Detailed reference viewed: 1 (0 ULg)
Bound or free: interaction of the C-terminal domain of Escherichia coli single-stranded DNA-binding protein (SSB) with the tetrameric core of SSB.
; ; et al
in Biochemistry (2014)Detailed reference viewed: 1 (0 ULg)
Engineering Specificity Changes on The RanBP2 Single-Stranded RNA Binding Zinc Finger.
Conference (2013, July 20)Detailed reference viewed: 7 (0 ULg)
A structural analysis of DNA binding by myelin transcription factor 1 double zinc fingers.
; ; et al
in Journal of Biological Chemistry (2013)Detailed reference viewed: 1 (0 ULg)
New insights into DNA recognition by zinc fingers revealed by structural analysis of the oncoprotein ZNF217
Vandevenne, Marylène ; ; et al
in Journal of Biological Chemistry (2013)Detailed reference viewed: 5 (2 ULg)
New insights into DNA recognition by zinc fingers revealed by structural analysis of the oncoprotein ZNF217 (Invited speaker from the Sydney Protein Group)
Conference (2012, September 25)Detailed reference viewed: 6 (0 ULg)
How to make good use of a “bad” enzyme: utilization of efficient β-lactamases for the benefits of biochemical research
Vandevenne, Marylène ; Galleni, Moreno ;
in Frère, Jean-Marie (Ed.) β-lactamases (2012)Detailed reference viewed: 18 (1 ULg)
Modular assembly of RanBP2-type zinc finger domains to target single- stranded RNA
; Vandevenne, Marylène ; et al
in Angewandte Chemie International Edition (2012)Detailed reference viewed: 2 (0 ULg)
Comparative functional analysis of the human macrophage chitotriosidase
Vandevenne, Marylène ; Campisi, Vincenzo ; Freichels, Astrid et al
in Protein Science : A Publication of the Protein Society (2011)Detailed reference viewed: 43 (1 ULg)
The multi-zinc finger protein ZNF217 contacts DNA through a two-finger domain.
; ; et al
in Journal of Biological Chemistry (2011)Detailed reference viewed: 2 (0 ULg)
Effects of monopropanediamino-beta-cyclodextrin on the denaturation process of the hybrid protein BlaPChBD.
Vandevenne, Marylène ; GASPARD, Genevieve ; 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 ▲]Detailed reference viewed: 67 (9 ULg)
The hybrid protein BlaPChBD as a versatile tool to study protein/ligand interactions.
Conference (2008, April 19)Detailed reference viewed: 4 (0 ULg)
Rapid and easy development of versatile tools to study protein/ligand interactions
Vandevenne, Marylène ; Gaspard, Gilles ; et al
in Protein engineering, design & selection (2008)Detailed reference viewed: 12 (0 ULg)