Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

BACKGROUND: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. RESULTS: A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 muM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway. CONCLUSIONS: The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules

Functionalized artificial bidomain proteins based on an α-solenoid protein repeat scaffold : a new class of artificial diels-alderases

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3' domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels-Alder (D-A) reaction. This allowed us to obtain four new artificial Diels-Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV-vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D-A reaction. They were found to be able to catalyze the enantioselective D-A reaction of azachalcone with cyclopentadiene with up to 38% yield and 52% enantiomeric excess, which validates the proposed strategy. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggest that artificial metalloenzymes based on bidomain A3-A3 proteins modified with nitrogen donor ligands may be suitable for further catalyst optimization and may constitute valuable tools toward more efficient and selective artificial biocatalysts

Specific GFP-binding artificial proteins ( Rep): a new tool for in vitro to live cell applications

International audienceA family of artificial proteins, named αRep, based on a natural family of helical repeat was previously designed. αRep members are efficiently expressed, folded and extremely stable proteins. A large αRep library was constructed creating proteins with a randomized interaction surface. In the present study, we show that the αRep library is an efficient source of tailor-made specific proteins with direct applications in biochemistry and cell biology. From this library, we selected by phage display αRep binders with nanomolar dissociation constants against the GFP. The structures of two independent αRep binders in complex with the GFP target were solved by X-ray crystallography revealing two totally different binding modes. The affinity of the selected αReps for GFP proved sufficient for practically useful applications such as pull-down experiments. αReps are disulfide free proteins and are efficiently and functionally expressed in eukaryotic cells: GFP-specific αReps are clearly sequestrated by their cognate target protein addressed to various cell compartments. These results suggest that αRep proteins with tailor-made specificity can be selected and used in living cells to track, modulate or interfere with intracellular processes

Interactions de cations métalliques avec les métallochaperonnes à cuivre Copz (enterococcus hirae) et Hah1 (humaine) (sélectivité, affinité et nature des complexes chaperonne-métal)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Nanoparticles Self-Assembly Driven by High Affinity Repeat Protein Pairing

International audienceProteins are the most specific yet versatile biological self-assembling agents with a rich chemistry. Nevertheless, the design of new proteins with recognition capacities is still in its infancy and has seldom been exploited for the self-assembly of functional inorganic nanoparticles. Here, we report on the protein-directed assembly of gold nanoparticles using purpose-designed artificial repeat proteins having a rigid but modular 3D architecture. αRep protein pairs are selected for their high mutual affinity from a library of 10(9) variants. Their conjugation onto gold nanoparticles drives the massive colloidal assembly of free-standing, one-particle thick films. When the average number of proteins per nanoparticle is lowered, the extent of self-assembly is limited to oligomeric particle clusters. Finally, we demonstrate that the aggregates are reversibly disassembled by an excess of one free protein. Our approach could be optimized for applications in biosensing, cell targeting, or functional nanomaterials engineerin

Analysis of the metal-binding selectivity of the metallochaperone CopZ from Enterococcus hirae by electrospray ionization mass spectrometry

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Metal-binding stoichiometry and selectivity of the copper chaperone CopZ from Enterococcus hirae

International audienc

pH sensitivity of YFPs is reduced upon AlphaRep binding: proof of concept in vitro and in living cells

International audienceYellow fluorescent proteins (YFPs) are commonly used in biology to track cellular processes, particularly as acceptors in experiments using the Förster Resonant Energy Transfer (FRET) phenomenon. However, their fluorescence intensity is strongly pH‐dependent, limiting their utility in acidic environments. Here, we explore the pH sensitivity of YFPs upon binding with an artificial repeat protein (αRep) both in vitro and in living cells. We show that αRep binds to Citrine, with high affinity in the nanomolar range at physiological and acidic pHs, leading to increased thermal stability of the complex. Moreover, αRep binding reduces Citrine's pKa by 0.75 pH units, leading to a decreased sensitivity to pH fluctuations. This effect can be generalized to other YFPs as Venus and EYFP in vitro. An efficient binding of αRep to Citrine has also been observed in living cells both at pH 7.4 and pH 6. This interaction leads to reduced variations of Citrine fluorescence intensity in response to pH variations in cells. Overall, the study highlights the potential of αReps as a tool to modulate the pH sensitivity of YFPs, paving the way for future exploration of biological events in acidic environments by FRET in combination with a pH‐insensitive cyan donor

Directed evolution of artificial repeat proteins as habit modifiers for the morphosynthesis of (111)-terminated gold nanocrystals

International audienceNatural biocomposites are shaped by proteins that have evolved to interact with inorganic materials. Protein directed evolution methods which mimic Darwinian evolution have proven highly successful to generate improved enzymes or therapeutic antibodies but have rarely been used to evolve protein-material interactions. Indeed, most reported studies have focused on short peptides and a wide range of oligopeptides with chemical binding affinity for inorganic materials have been uncovered by phage display methods. However, their small size and flexible unfolded structure prevent them from dictating the shape and crystallinity of the growing material. In the present work, a specific set of artificial repeat proteins (αRep), which exhibit highly stable 3D folding with a well-defined hypervariable interacting surface, is selected by directed evolution of a very efficient home-built protein library for their high and selective affinity for the Au(111) surface. The proteins are built from the extendable concatenation of self-compatible repeated motifs idealized from natural HEAT proteins. The high-yield synthesis of Au(111)-faceted nanostructures mediated by these αRep proteins demonstrates their chemical affinity and structural selectivity that endow them with high crystal habit modification performances. Importantly, we further exploit the protein shell spontaneously assembled on the nanocrystal facets to drive protein-mediated colloidal self-assembly and on-surface enzymatic catalysis. Our method constitutes a generic tool for producing nanocrystals with determined faceting, superior biocompatibility and versatile bio-functionalization towards plasmon-based devices and (bio)molecular sensors