RESUMEN
A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.
Asunto(s)
Cafeína , Citocromo P-450 CYP1A2 , Humanos , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Transporte de Electrón , Mutación , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/metabolismoRESUMEN
BACKGROUND: Physarum polycephalum is an unusual macroscopic myxomycete expressing a large range of glycosyl hydrolases. Among them, enzymes from the GH18 family can hydrolyze chitin, an important structural component of the cell walls in fungi and in the exoskeleton of insects and crustaceans. METHODS: Low stringency sequence signature search in transcriptomes was used to identify GH18 sequences related to chitinases. Identified sequences were expressed in E. coli and corresponding structures modelled. Synthetic substrates and in some cases colloidal chitin were used to characterize activities. RESULTS: Catalytically functional hits were sorted and their predicted structures compared. All share the TIM barrel structure of the GH18 chitinase catalytic domain, optionally fused to binding motifs, such as CBM50, CBM18, and CBM14, involved in sugar recognition. Assessment of the enzymatic activities following deletion of the C-terminal CBM14 domain of the most active clone evidenced a significant contribution of this extension to the chitinase activity. A classification based on module organization, functional and structural criteria of characterized enzymes was proposed. CONCLUSIONS: Physarum polycephalum sequences encompassing a chitinase like GH18 signature share a modular structure involving a structurally conserved catalytic TIM barrels decorated or not by a chitin insertion domain and optionally surrounded by additional sugar binding domains. One of them plays a clear role in enhancing activities toward natural chitin. GENERAL SIGNIFICANCE: Myxomycete enzymes are currently poorly characterized and constitute a potential source for new catalysts. Among them glycosyl hydrolases have a strong potential for valorization of industrial waste as well as in therapeutic field.
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Quitinasas , Mixomicetos , Physarum polycephalum , Quitinasas/genética , Quitinasas/química , Physarum polycephalum/metabolismo , Mixomicetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Quitina/química , AzúcaresRESUMEN
Conformational dynamics plays a critical role for the function of multidomain electron transfer complexes. While crystallographic or NMR approaches allow detailed insight into structures, lower resolution methods like cryo-electron microscopy can provide more information on dynamics. In silico structure modelling using AlphaFold was recently successfully extended to the prediction of protein complexes but its capability to address large conformational changes involved in catalysis remained obscure. We used bacterial CYP102A1 monooxygenase homodimer as a test case to design a competitive modelling approach (CMA) for assessing alternate conformations of multi-domain complexes. Predictions were confronted with published crystallographic and cryo-EM data, evidencing consistencies but also permitting some reinterpretation of experimental data. Structural determinants stabilising the new type of domain connectivity evidenced in this bacterial self-sufficient monooxygenase were analysed by CMA and used for in silico retro-engineering applied to its eukaryotic bi-component counterparts.
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Oxigenasas de Función Mixta , Catálisis , Microscopía por Crioelectrón , Transporte de Electrón , Conformación ProteicaRESUMEN
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
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Dosificación de Gen/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adaptación Fisiológica/genética , Aminoácidos Aromáticos/biosíntesis , Antibacterianos/farmacología , Doxiciclina/farmacología , Ingeniería Metabólica , Plásmidos/genéticaRESUMEN
Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents different in silico tools that were developed to uncover such channels in P450 crystal structures. It also lists some of the experimental evidence that actually suggest that these predicted channels might indeed play a critical role in modulating P450 functions. Amino acid residues at the entrance of the channels may participate to a first global ligand recognition of ligands by P450 enzymes before they reach the buried active site. Moreover, different P450 enzymes show different networks of predicted channels. The plasticity of P450 structures is also important to take into account when looking at how channels might play their role.
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Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Sitios de Unión , Sistema Enzimático del Citocromo P-450/química , Humanos , Hidrolasas/química , Hidrolasas/metabolismo , Simulación de Dinámica Molecular , Especificidad por SustratoRESUMEN
Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.
Asunto(s)
Citometría de Flujo/métodos , Proteínas Luminiscentes , Microfluídica/métodos , Escherichia coli/crecimiento & desarrollo , Biblioteca de Genes , Proteínas Fluorescentes Verdes , Metagenómica/métodos , Técnicas Analíticas Microfluídicas/métodos , Fenotipo , Saccharomyces cerevisiae/crecimiento & desarrollo , Coloración y Etiquetado/métodos , Proteína Fluorescente RojaRESUMEN
CcmK proteins are major constituents of icosahedral shells of ß-carboxysomes, a bacterial microcompartment that plays a key role for CO2 fixation in nature. Supported by the characterization of bidimensional (2D) layers of packed CcmK hexamers in crystal and electron microscopy structures, CcmK are assumed to be the major components of icosahedral flat facets. Here, we reassessed the validity of this model by studying CcmK isoforms from Synechocystis sp. PCC6803. Native mass spectrometry studies confirmed that CcmK are hexamers in solution. Interestingly, potential pre-assembled intermediates were also detected with CcmK2. Atomic-force microscopy (AFM) imaging under quasi-physiological conditions confirmed the formation of canonical flat sheets with CcmK4. Conversely, CcmK2 formed both canonical and striped-patterned patches, while CcmK1 assembled into remarkable supra-hexameric curved honeycomb-like mosaics. Mutational studies ascribed the propensity of CcmK1 to form round assemblies to a combination of two features shared by at least one CcmK isoform in most ß-cyanobacteria: a displacement of an α helical portion towards the hexamer edge, where a potential phosphate binding funnel forms between packed hexamers, and the presence of a short C-terminal extension in CcmK1. All-atom molecular dynamics supported a contribution of phosphate molecules sandwiched between hexamers to bend CcmK1 assemblies. Formation of supra-hexameric curved structures could be reproduced in coarse-grained simulations, provided that adhesion forces to the support were weak. Apart from uncovering unprecedented CcmK self-assembly features, our data suggest the possibility that transitions between curved and flat assemblies, following cargo maturation, could be important for the biogenesis of ß-carboxysomes, possibly also of other BMC.
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Proteínas Bacterianas/metabolismo , Silicatos de Aluminio/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cromatografía en Gel , Isomerismo , Espectrometría de Masas , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Mutación , Fosfatos/química , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Soluciones , Solventes/química , SynechocystisRESUMEN
BACKGROUND: Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. METHODS: A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. RESULTS: Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels. CONCLUSIONS: Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. GENERAL SIGNIFICANCE: The result provides a new basis for determining the structural elements that control functions in complex enzymes.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Esteroide Hidroxilasas/metabolismo , Animales , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Cumarinas/metabolismo , Ciclofosfamida/metabolismo , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Familia 2 del Citocromo P450 , Perros , Humanos , Cinética , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Esteroide Hidroxilasas/química , Esteroide Hidroxilasas/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
BACKGROUND: Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast. METHODS: CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone. RESULTS: CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6ß-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles. CONCLUSIONS: Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds. GENERAL SIGNIFICANCE: Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , Saccharomyces cerevisiae/genética , Pez Cebra/metabolismo , Animales , Benzo(a)pireno/metabolismo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.
Asunto(s)
Electrones , NADPH-Ferrihemoproteína Reductasa/química , Elasticidad , Flavinas/química , Humanos , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Soluciones , Rayos XRESUMEN
Fine tuning of individual enzyme expression level is necessary to alleviate metabolic imbalances in synthetic heterologous pathways. A known approach consists of choosing a suitable combination of promoters, based on their characterized strengths in model conditions. We questioned whether each step of a multiple-gene synthetic pathway could be independently tunable at the transcription level. Three open reading frames, coding for enzymes involved in a synthetic pathway, were combinatorially associated to different promoters on an episomal plasmid in Saccharomyces cerevisiae. We quantified the mRNA levels of the three genes in each strain of our generated combinatorial metabolic library. Our results evidenced that the ORF nature, position, and orientation induce strong discrepancies between the previously reported promoters' strengths and the observed ones. We conclude that, in the context of metabolic reconstruction, the strength of usual promoters can be dramatically affected by many factors. Among them, transcriptional interference and ORF nature seem to be predominant.
RESUMEN
BACKGROUND: A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. METHODS: Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. RESULTS: The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. CONCLUSIONS: Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. GENERAL SIGNIFICANCE: Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site.
Asunto(s)
Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Sitios de Unión , Catálisis , Citocromo P-450 CYP1A1/fisiología , Citocromo P-450 CYP1A2/fisiología , Simulación de Dinámica Molecular , Especificidad por SustratoRESUMEN
The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.
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Sistema Enzimático del Citocromo P-450/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Recombinantes de Fusión/metabolismo , Esteroides/análisis , Secuencia de Aminoácidos , Sistema Enzimático del Citocromo P-450/química , Humanos , Microsomas , Mifepristona , Modelos Moleculares , Datos de Secuencia Molecular , Sesquiterpenos Policíclicos , Proteínas Recombinantes de Fusión/química , Saccharomyces cerevisiae , Alineación de Secuencia , Sesquiterpenos , Esteroides/química , Esteroides/metabolismo , Especificidad por SustratoRESUMEN
Ethoxyresorufin is a classical substrate for vertebrate CYP1A enzymes. In Pterygoplichthys sp. (Loricariidae) this enzyme possesses 48 amino acids substitutions compared to CYP1A sequences from other vertebrate species. These substitutions or a certain subset substitution are responsible for the non-detection of the EROD reaction in this species liver microsomes. In the present study, we investigated the catalytic activity of Pterygoplichthys sp. CYP1A toward 15 potential substrates in order to understand the substrate preferences of this modified CYP1A. The fish gene was expressed in yeast and the accumulation of the protein was confirmed by both the characteristic P450-CO absorbance spectra and by detection with monoclonal antibodies. Catalytic activities were assayed with yeast microsomes and four resorufin ethers, six coumarin derivates, three flavones, resveratrol and ethoxyfluoresceinethylester. Results demonstrated that the initial velocity pattern of this enzyme for the resorufin derivatives is different from the one described for most vertebrate CYP1As. The initial velocity for the activity with the coumarin derivatives is several orders of magnitude higher than with the resorufins, i.e. the turnover number (kcat) for ECOD is 400× higher than for EROD. Nonetheless, the specificity constant (kcat/km) for EROD is only slightly higher than for ECOD. EFEE is degraded at a rate comparable to the resorufins. Pterygoplichthys sp. CYP1A also degrades 7-methoxyflavone and ß-naphthoflavone but not resveratrol and chrysin. These results indicate a divergent substrate preference for Pterygoplichthys sp. CYP1A, which may be involved in the adaptation of Loricariidae fish to their particular environment and feeding habits.
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Bagres/fisiología , Citocromo P-450 CYP1A1/metabolismo , Contaminantes Químicos del Agua/metabolismo , Animales , Citocromo P-450 CYP1A1/genética , Regulación Enzimológica de la Expresión Génica , Microsomas Hepáticos/enzimología , Saccharomyces cerevisiae/genética , Especificidad por Sustrato/fisiologíaRESUMEN
Alternative pathways of metabolic networks represent the escape routes that can reduce drug efficacy and can cause severe adverse effects. In this paper we introduce a mathematical algorithm and a coding system for rapid computational construction of metabolic networks. The initial data for the algorithm are the source substrate code and the enzyme/metabolite interaction tables. The major strength of the algorithm is the adaptive coding system of the enzyme-substrate interactions. A reverse application of the algorithm is also possible, when optimisation algorithm is used to compute the enzyme/metabolite rules from the reference network structure. The coding system is user-defined and must be adapted to the studied problem. The algorithm is most effective for computation of networks that consist of metabolites with similar molecular structures. The computation of the cholesterol biosynthesis metabolic network suggests that 89 intermediates can theoretically be formed between lanosterol and cholesterol, only 20 are presently considered as cholesterol intermediates. Alternative metabolites may represent links with other metabolic networks both as precursors and metabolites of cholesterol. A possible cholesterol-by-pass pathway to bile acids metabolism through cholestanol is suggested.
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Algoritmos , Colesterol/biosíntesis , Biología Computacional/métodos , Modelos Biológicos , Colesterol/química , Simulación por Computador , Lanosterol/química , Lanosterol/metabolismo , Redes y Vías Metabólicas , Modelos MolecularesRESUMEN
BACKGROUND: A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs - complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes. METHODS: An original algorithmic approach (Global Sequence Signature-GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences. RESULTS: Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation. CONCLUSIONS: Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. GENERAL SIGNIFICANCE: In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.
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Algoritmos , Camélidos del Nuevo Mundo/genética , Camelus/genética , Regiones Determinantes de Complementariedad/genética , Cadenas Pesadas de Inmunoglobulina/genética , Análisis de Secuencia de Proteína/métodos , Animales , Camélidos del Nuevo Mundo/inmunología , Camelus/inmunología , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Estructura Secundaria de Proteína , Alineación de Secuencia/métodosRESUMEN
Synthetic proteo-nucleic structures (PDNAs) encompassing a single-stranded DNA sequence covalently attached to a redox protein domain able to interact with surface or matrix were designed and characterized. They constitute versatile building blocks alternative to regular DNA for creating scaffolds with optical, electrical, or catalytic properties. PDNAs self-assemble in the presence of complementary oligonucleotides, to form a network of protein domains linked by double-stranded DNA segments. Electrophoretic and hydrodynamic behaviors of PDNAs and corresponding DNA were compared under electrophoresis and gel filtration conditions. Hybridization rates between small and large assemblies were characterized by rapid-mixing experiments. Results showed that the protein part significantly contributes to hydrodynamic behaviors of structures but marginally affects the conformation and hybridization properties of the nucleic domain. PDNA metal-mediated complexes with nitriloacetate-modified phospholipids can diffuse and interact at the surface of vesicles or supported membranes. Surface plasmon resonance analysis of membrane-PDNA interactions indicated that two protein units are required to allow stable surface association and that surface occupancy constrains assembly sizes. High-speed atomic force microscopy illustrated rapid lateral diffusion of assemblies on mica, revealing transient association between noncomplementary PDNA extremities and frequent trapping by surface defects. Regularly organized protein domains were visualized using a larger DNA framework.
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ADN/química , Proteínas/química , Silicatos de Aluminio , Cromatografía en Gel , Citocromos b/química , Metales/química , Microscopía de Fuerza Atómica , Estructura Molecular , Soluciones , Resonancia por Plasmón de SuperficieRESUMEN
BACKGROUND: Quantitative modeling of the self-assembly of DNA tiles leading either to defined end-products or distribution of biopolymers is of practical importance for biotechnology and synthetic biology. METHODS: The combinatorial process describing tile assembly was implemented into a generic algorithm allowing quantitative description of the population of significant species accumulating during the reaction course. Experimental formation and characterization by optical and electrophoresis approaches of copolymers resulting from the self-assembly of a limited number of half-complementary tiles were used to define and validate generic rules allowing definition of model parameters. RESULTS: Factors controlling the structure and the dynamic of the oligomer population were evidenced for assemblies leading or not to defined end-products. Primary parameters were experimentally determined using rapid mixing experiments. Adjustment of simulations to experimental profiles allowed definition of generic rules for calculation of secondary parameters that take into account macro- and microenvironment of individual hybridization steps. In the case of copolymers, accurate simulation of experimental profiles was achieved for formation of linear assemblies. CONCLUSIONS: Overall length of species and structure of the DNA regions flanking the hybridization sites are critical parameters for which calculation rules were defined. The computational approach quantitatively predicted the parameters affecting time-course and distribution of accumulating products for different experimental designs. GENERAL SIGNIFICANCE: The computational and parameter evaluation procedures designed for the assembly of DNA tiles into large 1D-structures are more generally applicable for the construction of non-DNA polymers by extremities-specific recognition of molecular blocks.
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ADN/química , Modelos Genéticos , Secuencia de Bases , Técnicas Químicas Combinatorias , Simulación por Computador , ADN/genética , Diseño de Fármacos , Cinética , Datos de Secuencia Molecular , Reproducibilidad de los ResultadosRESUMEN
CPR (NADPH-cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.