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1.
Chembiochem ; : e202400139, 2024 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-38682718

RESUMO

A binuclear Cu(II) cofactor was covalently bound to a lauric acid anchor. The resulting conjugate was characterized then combined with beta-lactoglobulin (ßLG) to generate a new biohybrid following the so-called "Trojan horse" strategy. This biohybrid was examined for its effectiveness in the oxidation of a catechol derivative to the corresponding quinone. The resulting biohybrid did not exhibit the sought after catecholase activity, likely due to its ability to bind and stabilize the semiquinone radical intermediate DTB-SQ. This semi-quinone radical was stabilized only in the presence of the protein and was characterized using optical and magnetic spectroscopic techniques, demonstrating stability for over 16 hours. Molecular docking studies revealed that this stabilization could occur owing to interactions of the semi-quinone with hydrophobic amino acid residues of ßLG.

2.
J Struct Biol ; 215(3): 108012, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37567372

RESUMO

Synthetic ɑRep repeat proteins are engineered as Brick and Staple protein pairs that together self-assemble into helical filaments. In most cases, the filaments spontaneously form supercrystals. Here, we describe an expanded series of ɑRep Bricks designed to stabilize the interaction between consecutive Bricks, to control the length of the assembled multimers, or to alter the spatial distribution of the Staple on the filaments. The effects of these Brick modifications on the assembly, on the final filament structure and on the crystal symmetry are analyzed by biochemical methods, electron microscopy and small angle X-ray scattering. We further extend the concept of Brick/Staple protein origami by designing a new type of "Janus"-like Brick protein that is equally assembled by orthogonal staples binding its inner or outer surfaces and thus ending inside or outside the filaments. The relative roles of longitudinal and lateral associations in the assembly process are discussed. This set of results demonstrates important proofs-of-principle for engineering these remarkably versatile proteins toward nanometer-to-micron scale constructions.


Assuntos
Citoesqueleto , Proteínas , Proteínas/genética , Proteínas/química , Microscopia Eletrônica
3.
Proc Natl Acad Sci U S A ; 120(11): e2218428120, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36893280

RESUMO

A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.


Assuntos
Nanoestruturas , Proteínas , Difração de Raios X , Espalhamento a Baixo Ângulo , Proteínas/química , Temperatura , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Conformação de Ácido Nucleico
4.
Int J Mol Sci ; 23(23)2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36498969

RESUMO

The covalent insertion of a cobalt heme into the cavity of an artificial protein named alpha Rep (αRep) leads to an artificial cobalt hemoprotein that is active as a catalyst not only for the photo-induced production of H2, but also for the reduction of CO2 in a neutral aqueous solution. This new artificial metalloenzyme has been purified and characterized by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), circular dichroism, and UltraViolet-Visible spectroscopy. Using theoretical experiments, the structure of this biohybrid and the positioning of the residues near the metal complex were examined, which made it possible to complete the coordination of the cobalt ion by an axial glutamine Gln283 ligand. While the Co(III)-porphyrin catalyst alone showed weak catalytic activity for both reactions, 10 times more H2 and four times more CO2 were produced when the Co(III)-porphyrin complex was buried in the hydrophobic cavity of the protein. This study thus provides a solid basis for further improvement of these biohybrids using well-designed modifications of the second and outer coordination sphere by site-directed mutagenesis of the host protein.


Assuntos
Complexos de Coordenação , Hemeproteínas , Porfirinas , Cobalto/química , Dióxido de Carbono/química , Complexos de Coordenação/química , Catálise , Hidrogênio/química
5.
PLoS Pathog ; 18(9): e1010799, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36067253

RESUMO

The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.


Assuntos
Enzima de Conversão de Angiotensina 2 , Tratamento Farmacológico da COVID-19 , Proteínas Recombinantes de Fusão , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Antivirais/farmacologia , Humanos , Peptidil Dipeptidase A/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
Proc Natl Acad Sci U S A ; 119(19): e2120098119, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35507869

RESUMO

Microtubule dynamics is regulated by various cellular proteins and perturbed by small-molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin ß subunit. Moreover, a PN2-3 N-terminal stretch lies in a ß-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small-molecule inhibitors of microtubule assembly.


Assuntos
Tubulina (Proteína) , Vinca , Microtúbulos/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina , Vinca/metabolismo
7.
Biochemistry ; 59(48): 4591-4600, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33231438

RESUMO

The selective targeting of protein-protein interactions remains a significant determinant for the proper modulation and regulation of cell apoptosis. Prototypic galectins such as human galectin-7 (GAL-7) are characterized by their ability to form homodimers that control the molecular fate of a cell by mediating subtle yet critical glycan-dependent interactions between pro- and anti-apoptotic molecular partners. Altering the structural architecture of GAL-7 can therefore result in resistance to apoptosis in various human cancer cells, further illustrating its importance in cell survival. In this study, we used a combination of biophysical and cellular assays to illustrate that binding of a water-soluble meso-tetraarylporphyrin molecule to GAL-7 induces protein oligomerization and modulation of GAL-7-induced apoptosis in human Jurkat T cells. Our results suggest that the integrity of the GAL-7 homodimer architecture is essential for its molecular function, in addition to providing an interesting porphyrin binding modulator for controlling apoptosis in mammalian cells.


Assuntos
Galectinas/química , Galectinas/metabolismo , Mesoporfirinas/química , Mesoporfirinas/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Galectinas/farmacologia , Humanos , Técnicas In Vitro , Células Jurkat , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Espalhamento a Baixo Ângulo , Solubilidade , Difração de Raios X
8.
Chemistry ; 26(65): 14929-14937, 2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-32588931

RESUMO

A novel inducible artificial metalloenzyme obtained by covalent attachment of a manganese(III)-tetraphenylporphyrin (MnTPP) to the artificial bidomain repeat protein, (A3A3')Y26C, is reported. The protein is part of the αRep family. The biohybrid was fully characterized by MALDI-ToF mass spectrometry, circular dichroism and UV/Vis spectroscopies. The peroxidase and monooxygenase activities were evaluated on the original and modified scaffolds including those that have a) an additional imidazole, b) a specific αRep bA3-2 that is known to induce the opening of the (A3A3') interdomain region and c) a derivative of the αRep bA3-2 inducer extended with a His6 -Tag (His6 -bA3-2). Catalytic profiles are highly dependent on the presence of co-catalysts with the best activity obtained with His6 -bA3-2. The entire mechanism was rationalized by an integrative molecular modeling study that includes protein-ligand docking and large-scale molecular dynamics. This constitutes the first example of an entirely artificial metalloenzyme with inducible peroxidase and monooxygenase activities, reminiscent of allosteric regulation of natural enzymatic pathways.


Assuntos
Oxigenases de Função Mista/metabolismo , Catálise , Metaloproteínas , Peroxidases
9.
ACS Nano ; 14(5): 5956-5967, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32216328

RESUMO

Although antibodies remain a primary recognition element in all forms of biosensing, functional limitations arising from their size, stability, and structure have motivated the development and production of many different artificial scaffold proteins for biological recognition. However, implementing such artificial binders into functional high-performance biosensors remains a challenging task. Here, we present the design and application of Förster resonance energy transfer (FRET) nanoprobes comprising small artificial proteins (αRep bidomains) labeled with a Tb complex (Tb) donor on the C-terminus and a semiconductor quantum dot (QD) acceptor on the N-terminus. Specific binding of one or two protein targets to the αReps induced a conformational change that could be detected by time-resolved Tb-to-QD FRET. These single-probe FRET switches were used in a separation-free solution-phase assay to quantify different protein targets at sub-nanomolar concentrations and to measure the conformational changes with sub-nanometer resolution. Probing ligand-receptor binding under physiological conditions at very low concentrations in solution is a special feature of FRET that can be efficiently combined with other structural characterization methods to develop, understand, and optimize artificial biosensors. Our results suggest that the αRep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live-cell imaging of ligand-receptor interactions.


Assuntos
Técnicas Biossensoriais , Pontos Quânticos , Transferência Ressonante de Energia de Fluorescência , Semicondutores , Térbio
10.
Nanoscale ; 12(7): 4612-4621, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-32043516

RESUMO

Hybrid nanostructures are constructed by the direct coupling of fluorescent quantum dots and plasmonic gold nanoparticles. Self-assembly is directed by the strong affinity between two artificial α-repeat proteins that are introduced in the capping layers of the nanoparticles at a controlled surface density. The proteins have been engineered to exhibit a high mutual affinity, corresponding to a dissociation constant in the nanomolar range, towards the protein-functionalized quantum dots and gold nanoparticles. Protein-mediated self-assembly is evidenced by surface plasmon resonance and gel electrophoresis. The size and the structure of colloidal superstructures of complementary nanoparticles are analyzed by transmission electron microscopy and small angle X-ray scattering. The size of the superstructures is determined by the number of proteins per nanoparticle. The well-defined geometry of the rigid protein complex sets a highly uniform interparticle distance of 8 nm that affects the emission properties of the quantum dots in the hybrid ensembles. Our results open the route to the design of hybrid emitter-plasmon colloidal assemblies with controlled near-field coupling and better optical response.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Proteínas/química , Pontos Quânticos/química , Ressonância de Plasmônio de Superfície , Eletroforese
11.
Nanoscale ; 11(37): 17485-17497, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31532442

RESUMO

Natural 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.


Assuntos
Evolução Molecular Direcionada , Ouro/química , Nanopartículas Metálicas/química , Nanopartículas/química , Biblioteca de Peptídeos
12.
Proc Natl Acad Sci U S A ; 116(20): 9859-9864, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31036638

RESUMO

Nucleation is one of the least understood steps of microtubule dynamics. It is a kinetically unfavorable process that is templated in the cell by the γ-tubulin ring complex or by preexisting microtubules; it also occurs in vitro from pure tubulin. Here we study the nucleation inhibition potency of natural or artificial proteins in connection with their binding mode to the longitudinal surface of α- or ß-tubulin. The structure of tubulin-bound CopN, a Chlamydia protein that delays nucleation, suggests that this protein may interfere with two protofilaments at the (+) end of a nucleus. Designed ankyrin repeat proteins that share a binding mode similar to that of CopN also impede nucleation, whereas those that target only one protofilament do not. In addition, an αRep protein predicted to target two protofilaments at the (-) end does not delay nucleation, pointing to different behaviors at both ends of the nucleus. Our results link the interference with protofilaments at the (+) end and the inhibition of nucleation.


Assuntos
Proteínas de Bactérias/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Chlamydophila pneumoniae
13.
Sci Rep ; 9(1): 1178, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718544

RESUMO

Artificial proteins binding any predefined "target" protein can now be efficiently generated using combinatorial libraries based on robust protein scaffolds. αRep is such a family of artificial proteins, based on an α-solenoid protein repeat scaffold. The low aggregation propensity of the specific "binders" generated from this library opens new protein engineering opportunities such as the creation of biosensors within multidomain constructions. Here, we have explored the properties of two new types of artificial bidomain proteins based on αRep structures. Their structural and functional properties are characterized in detail using biophysical methods. The results clearly show that both bidomain proteins adopt a closed bivalve shell-like conformation, in the ligand free form. However, the presence of ligands induces a conformational transition, and the proteins adopt an open form in which each domain can bind its cognate protein partner. The open/closed equilibria alter the affinities of each domain and induce new cooperative effects. The binding-induced relative domain motion was monitored by FRET. Crystal structures of the chimeric proteins indicate that the conformation of each constituting domain is conserved but that their mutual interactions explain the emergent properties of these artificial bidomain proteins. The ligand-induced structural transition observed in these bidomain proteins should be transferable to other αRep proteins with different specificity and could provide the basis of a new generic biosensor design.


Assuntos
Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Fenômenos Biofísicos , Cristalografia por Raios X , Ligantes , Ligação Proteica , Proteínas Recombinantes/genética
14.
Structure ; 27(3): 497-506.e4, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30661854

RESUMO

Microtubules are cytoskeletal filaments of eukaryotic cells made of αß-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin. Turbidity experiments indicate that these αReps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (-) end. Structural data indicate that they do so by targeting the α-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the α subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These α-tubulin-specific αReps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation.


Assuntos
Proteínas Recombinantes de Fusão/farmacologia , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Moleculares , Biblioteca de Peptídeos , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Sequências Repetitivas de Aminoácidos
15.
J Struct Biol ; 201(2): 88-99, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28823563

RESUMO

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas de Bactérias/química , Cristalização/métodos , Ensaio de Imunoadsorção Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Histidina Quinase/química , Biblioteca de Peptídeos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/genética , Sequências Repetitivas de Aminoácidos , Tetra-Hidrofolato Desidrogenase/química
16.
Front Microbiol ; 9: 3014, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30671027

RESUMO

YabT is a serine/threonine kinase of the Hanks family from Bacillus subtilis, which lacks the canonical extracellular signal receptor domain but is anchored to the membrane through a C-terminal transmembrane helix. A previous study demonstrated that a basic juxtamembrane region corresponds to a DNA-binding motif essential for the activation of YabT trans-autophosphorylation. YabT is expressed during spore development and localizes to the asymmetric septum where it specifically phosphorylates essential proteins involved in genome maintenance, such as RecA, SsbA, and YabA. YabT has also been shown to phosphorylate proteins involved in protein synthesis, such as AbrB and Ef-Tu, suggesting a possible regulatory role in the progressive metabolic quiescence of the forespore. Finally, cross phosphorylations with other protein kinases implicate YabT in the regulation of numerous other cellular processes. Using an artificial protein scaffold as crystallization helper, we determined the first crystal structure of this DNA-dependent bacterial protein kinase. This allowed us to trap the active conformation of the kinase domain of YabT. Using NMR, we showed that the basic juxtamembrane region of YabT is disordered in the absence of DNA in solution, just like it is in the crystal, and that it is stabilized upon DNA binding. In comparison with its closest structural homolog, the mycobacterial kinase PknB allowed us to discuss the dimerization mode of YabT. Together with phosphorylation assays and DNA-binding experiments, this structural analysis helped us to gain new insights into the regulatory activation mechanism of YabT.

17.
Sci Rep ; 7(1): 16335, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-29180782

RESUMO

A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (αRep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of αRep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions. Two strong αRep binders were isolated from the screen, αRep4E3 (32 kDa; 7 internal repeats) and αRep9A8 (28 kDa; 6 internal repeats). Their antiviral activity against HIV-1 was evaluated in VLP-producer cells and in human SupT1 cells challenged with HIV-1. Both αRep4E3 and αRep9A8 showed a modest but significant antiviral effects in all bioassays and cell systems tested. They did not prevent the proviral integration reaction, but negatively interfered with late steps of the HIV-1 life cycle: αRep4E3 blocked the viral genome packaging, whereas αRep9A8 altered both virus maturation and genome packaging. Interestingly, SupT1 cells stably expressing αRep9A8 acquired long-term resistance to HIV-1, implying that αRep proteins can act as antiviral restriction-like factors.


Assuntos
Proteínas de Transporte/metabolismo , Produtos do Gene gag/metabolismo , Genoma Viral , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Nucleocapsídeo/metabolismo , Montagem de Vírus , Animais , Proteínas do Capsídeo/metabolismo , Proteínas de Transporte/química , Linhagem Celular , Humanos , Modelos Biológicos , Conformação Proteica em alfa-Hélice , Linfócitos T/metabolismo , Linfócitos T/virologia , Replicação Viral
18.
Chemistry ; 23(42): 10156-10166, 2017 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-28543753

RESUMO

αRep refers to a new family of artificial proteins based on a thermostable α-helical repeated motif. One of its members, αRep A3, forms a stable homo-dimer with a wide cleft that is able to accommodate metal complexes and thus appears to be suitable for generating new artificial biocatalysts. Based on the crystal structure of αRep A3, two positions (F119 and Y26) were chosen, and independently changed into cysteine residues. A phenanthroline ligand was covalently attached to the unique cysteine residue of each protein variant, and the corresponding biohybrids were purified and characterized. Once mutated and coupled to phenanthroline, the protein remained folded and dimeric. Copper(II) was specifically bound by the two biohybrids with two different binding modes. Furthermore, the holo-biohybrid A3F119NPH was found to be capable of enantioselectively catalyzing Diels-Alder (D-A) cycloadditions with up to 62 % ee. This study validates the choice of the αRep A3 dimer as a protein scaffold and provides a promising new route for the design and production of new enantioselective biohybrids based on entirely artificial proteins obtained from a highly diverse library.


Assuntos
Metaloproteínas/metabolismo , Sequência de Aminoácidos , Catálise , Dicroísmo Circular , Cobre/química , Reação de Cicloadição , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica , Espectrometria de Massas , Metaloproteínas/química , Fenantrolinas/química , Dobramento de Proteína , Alinhamento de Sequência , Estereoisomerismo
19.
J Struct Biol ; 195(1): 19-30, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27181418

RESUMO

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 (αßα) 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.


Assuntos
Simulação por Computador/normas , Evolução Molecular Direcionada/métodos , Proteínas/química , Proteínas Recombinantes/química , Cristalografia por Raios X , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína
20.
ACS Nano ; 10(3): 3176-85, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26863288

RESUMO

Proteins 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 engineering.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Proteínas/química , Nanopartículas Metálicas/ultraestrutura , Modelos Moleculares , Nanotecnologia/métodos
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