RESUMO
Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range.
Assuntos
Placofilinas/química , Naftalenossulfonato de Anilina/metabolismo , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Difusão Dinâmica da Luz , Humanos , Concentração de Íons de Hidrogênio , Placofilinas/isolamento & purificação , Conformação Proteica , Desnaturação Proteica , Domínios Proteicos , Soluções , Espectrometria de Fluorescência , UltracentrifugaçãoRESUMO
The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family (ß-α-ß-ß-ß-α) at its N-terminal region (residues 1100), whereas the C terminus (residues 100197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.
Assuntos
Proteínas de Bactérias/química , Proteínas Ribossômicas/química , Ribossomos/química , Synechocystis/química , Motivos de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Soluções , Synechocystis/metabolismo , TermodinâmicaRESUMO
The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain.
Assuntos
Proteínas de Membrana/química , Proteínas Mitocondriais/química , Multimerização Proteica , Estrutura Secundária de Proteína , Soluções/química , Sequência de Aminoácidos , Dicroísmo Circular , Simulação por Computador , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Dodecilsulfato de Sódio/químicaRESUMO
We propose a multiscale protocol for the simulation of conformation and dynamics of dendrimer molecules in dilute solution. Conformational properties (radius of gyration, mass distribution, and scattering intensities) and overall hydrodynamic properties (translational diffusion and intrinsic viscosity) are predicted by means of a very simple coarse-grained bead-and-spring model, whose parameters are not adjusted against experimental properties, but rather they are obtained from previous, atomic-level simulations which are also quite simple, performed with small fragments and Langevin dynamics simulation. The scheme is described and applied systematically to four different dendrimer molecules with up to seven generations. The predictive capability of this scheme is tested by comparison with experimental data. It is found that the predicted geometric and hydrodynamic radii of the dendrimer molecules are in agreement (typical error is about 4%) with a large set experimental values of the four dendrimers with various numbers of generations. Agreement with some X-ray scattering experimental intensities also confirms the good prediction of the internal structure. This scheme is easily extendable to study more complex molecules (e.g., functionalized dendrimers) and to simulate internal dynamics.
RESUMO
The formation of associative networks in semidilute aqueous solutions of hydrophobically modified hydroxyethylcellulose (HM-HEC) is dependent on intermolecular hydrophobic interactions. Addition of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) monomers to the system provides decoupling of these associations via inclusion complex formation with the polymer hydrophobic tails. Results from viscosity, polymer NMR self-diffusion, and dynamic light scattering (DLS) measurements show that the hydrophobic interactions in HM-HEC solutions are effectively suppressed when the level of HP-beta-CD addition increases. Small-angle neutron scattering (SANS) results reveal that the large-scale association complexes in HM-HEC solutions are strongly diminished when the concentration of HP-beta-CD rises. The time correlation data obtained from the DLS experiments unveiled the existence of two relaxation modes: one single exponential at short times followed by a stretched exponential at longer times. The fast mode is always diffusive, whereas the slow mode exhibits progressively stronger wavevector dependence as the intensity of the hydrophobic interactions increases. This feature, as well as the accompanying drop of the stretched exponential beta as the HP-beta-CD concentration decreases, is attributed to enhanced hydrophobic interactions and can be well rationalized in the framework of the coupling model of Ngai.
RESUMO
Sulfatases catalyze hydrolysis of sulfate groups. They have a key role in regulating the sulfation states that determine the function of several scaffold molecules. Currently, there are no studies of the conformational stability of endosulfatases. In this work, we describe the structural features and conformational stability of a 4-O-endosulfatase (EndoV) from a marine bacterium, which removes specifically the 4-O-sulfate from chondroitin sulfate/dermatan sulfate. For that purpose, we have used several biophysical techniques, namely, fluorescence, circular dichroism (CD), FTIR spectroscopy, analytical ultracentrifugation (AUC), differential scanning calorimetry (DSC), mass spectrometry (MS), dynamic light scattering (DLS) and size exclusion chromatography (SEC). The protein was a dimer with an elongated shape. EndoV acquired a native-like structure in a narrow pH range (7.0-9.0); it is within this range where the protein shows the maximum of enzymatic activity. The dimerization did not involve the presence of disulphide-bridges as suggested by AUC, SEC and DLS experiments in the presence of ß-mercaptoethanol (ß-ME). EndoV secondary structure is formed by a mixture of α and ß-sheet topology, as judged by deconvolution of CD and FTIR spectra. Thermal and chemical denaturations showed irreversibility and the former indicates that protein did not unfold completely during heating.
Assuntos
Proteínas de Bactérias/metabolismo , Sulfatos de Condroitina/metabolismo , Condroitinases e Condroitina Liases/metabolismo , Dermatan Sulfato/análogos & derivados , Vibrio/enzimologia , Proteínas de Bactérias/química , Biocatálise , Fenômenos Biofísicos , Varredura Diferencial de Calorimetria , Condroitinases e Condroitina Liases/química , Cromatografia em Gel , Dicroísmo Circular , Dermatan Sulfato/metabolismo , Concentração de Íons de Hidrogênio , Desnaturação Proteica , Multimerização Proteica , Desdobramento de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Sulfatos/metabolismo , TemperaturaRESUMO
The phosphotransferase system (PTS) is involved in the use of carbon sources in bacteria. Bacillus sphaericus, a bacterium with the ability to produce insecticidal proteins, is unable to use hexoses and pentoses as the sole carbon source, but it has ptsHI genes encoding the two general proteins of the PTS: enzyme I (EI) and the histidine phosphocarrier (HPr). In this work, we describe the biophysical and structural properties of HPr from B. sphaericus, HPr(bs), and its affinity towards EI of other species to find out whether there is inter-species binding. Conversely to what happens to other members of the HPr family, HPr(bs) forms several self-associated species. The conformational stability of the protein is low, and it unfolds irreversibly during heating. The protein binds to the N-terminal domain of EI from Streptomyces coelicolor, EIN(sc), with a higher affinity than that of the natural partner of EIN(sc), HPr(sc). Modelling of the complex between EIN(sc) and HPr(bs) suggests that binding occurs similarly to that observed in other HPr species. We discuss the functional implications of the oligomeric states of HPr(bs) for the glycolytic activity of B. sphaericus, as well as a strategy to inhibit binding between HPr(sc) and EIN(sc).
Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Proteínas de Bactérias/química , Calorimetria , Histidina , Temperatura Alta , Hidrodinâmica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peptídeos , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Fosforilação , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Streptomyces coelicolor/metabolismo , TermodinâmicaRESUMO
The calculation of solution properties of flexible macromolecules and other nanoparticles requires, in addition to the hydrodynamic formalisms needed for the sedimentation coefficient and other transport properties, the consideration of the conformational statistics and internal dynamics. The latter aspects can be handled with simulation methods like Monte Carlo and Brownian dynamics. An example of a Monte Carlo simulation for a model specific of DNA is illustrated with results for the several solution properties over an extremely wide range of molecular weight. The convenience of having computational tools of a quite general applicability has prompted us to implement the simulation and hydrodynamic treatments in software packages, MONTEHYDRO for Monte Carlo, and SIMUFLEX for Brownian dynamics which-with a scope similar to the HYDRO suite for rigid particles-can handle a variety of situations. As an application of the new methodology to a yet unclear problem in analytical ultracentrifugation, in a simple application of the SIMUFLEX software, we present a simulation of the so-called anomalous sedimentation of very long DNA molecules, obtaining results for the experimentally observable rotor-speed-dependence of the sedimentation coefficient.
Assuntos
Hidrodinâmica , Substâncias Macromoleculares/análise , Soluções/química , Ultracentrifugação/instrumentação , Ultracentrifugação/métodos , Simulação por Computador , MaleabilidadeRESUMO
This review elucidates several aspects on the behavior of charged polysaccharides and mucin. Viscosification of dilute aqueous solutions of hyaluronan (HA) occurs in the course of time at low shear flow, whereas shear thinning as time evolves is found at moderate shear rates. Hydrogen bonds and electrostatic interaction play an important role for the emergence of these features. No time effect of the viscosity is observed for semidilute HA solutions. A degradation of HA is observed at low and high pH and this effect continues over long times, and it is only in the approximate interval 5
Assuntos
Ácido Hialurônico/química , Mucinas/química , Polissacarídeos/química , Simulação por Computador , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Método de Monte Carlo , Concentração Osmolar , Resistência ao Cisalhamento , Eletricidade Estática , Tensoativos/química , ViscosidadeRESUMO
A bead-and-spring model has been used to simulate the behavior of thermoresponsive asymmetric diblock amphiphilic copolymers with aid of Monte Carlo simulations. The alteration of the thermodynamic conditions was mimicked by using a Lennard-Jones potential, which was related to the measured temperatures by comparison with experimental data for aqueous solutions of two sets of diblock copolymers, namely methoxypoly(ethylene glycol)-block-poly(N-isopropylacrylamide), one with different lengths of the hydrophilic block (MPEG(n)-b-PNIPAAM(71)) and one with varying lengths of the hydrophobic block (MPEG(57)-b-PNIPAAM(m)). The influence of the length of both the thermoresponsive and the hydrophilic block on the size and conformation of single molecules at various temperatures was studied by means of simulations. The temperature-induced contraction of the copolymer (MPEG(n)-b-PNIPAAM(71)) entities is only modestly affected by changing the length of the hydrophilic block, whereas for the MPEG(57)-b-PNIPAAM(m) copolymer both the transition temperature and the magnitude of the compression of the molecules are strongly influenced by the length of the thermosensitive block. When the MPEG chain fully covers the hydrophobic core, the copolymer moieties are stabilized, whereas poorly covered cores can promote interchain aggregation at elevated temperatures.
Assuntos
Acrilamidas/química , Interações Hidrofóbicas e Hidrofílicas , Polietilenoglicóis/química , Temperatura , Modelos Moleculares , Conformação Molecular , Método de Monte Carlo , Soluções , TermodinâmicaRESUMO
A computer programs suite, SIMUFLEX, has been constructed for the calculation of solution properties of flexible macromolecules modeled as bead-and-connector models of arbitrary topology. The suite consists mainly of two independent programs, BROWFLEX that generates the macromolecular trajectory by using the Brownian dynamics technique and ANAFLEX that analyzes that trajectory to get solution properties of the macromolecule. In this paper, we describe theoretical aspects about the macromolecular model and the Brownian dynamics algorithm used and describe some of the numerous properties that can be evaluated. In order to provide examples of the application of the methodology, we present simulations of dynamic properties of DNA with length ranging from 10 to 10(5) base pairs. SIMUFLEX is able to run simulations with more or less coarse-grained models, thus enabling such multiple-scale studies.