RESUMO
Monocrystals of dinuclear µ-1,4-bis(3-aminopropyl)piperazine-κ4N1,N1':N4,N4'-bis[bis(tri-tert-butoxysilanethiolato-κS)cadmium(II)], [Cd2(C12H27O3SSi)4(C10H24N4)] or [Cd2{SSi(OtBu)3}4(µ-BAPP)], 1, and polynuclear catena-poly[[bis(tri-tert-butoxysilanethiolato-κS)cadmium(II)]-µ-1,4-bis(3-aminopropyl)piperazine-κ2N1':N4'], [Cd(C12H27O3SSi)2(C10H24N4)]n or [Cd{SSi(OtBu)3}2(µ-BAPP)]n, 2, with 1,4-bis(3-aminopropyl)piperazine (BAPP) and tri-tert-butoxysilanethiolate ligands, were obtained from the same ratio of reactants, but with different solvents used for the crystallization processes. The structures and properties of both complexes were characterized using elemental analysis, X-ray diffraction and FT-IR, 1H NMR and luminescence spectroscopy. Applied density functional theory (DFT) computational methods and noncovalent interaction (NCI) analysis were used for geometry optimization and visualization of the interactions between the metallic centres and their surroundings. The X-ray analysis revealed four-coordinate CdII centres bound to two S atoms of the silanethiolate groups and two N atoms of the BAPP ligand; however, it chelates to tertiary and primary N atoms in 1, whilst in 2 it does not chelate and bonds only to RNH2. The photoluminescence properties of complexes 1 and 2 result from free-ligand emission and differ significantly from each other with respect to emission intensity. Additionally, antifungal activity was investigated against 18 isolates of fungi. Compound 1 strongly inhibited the growth of three dermatophytes: Epidermophyton floccosum, Microsporum canis and Trichophyton rubrum.
RESUMO
In this work, we investigated the influence of stabilizing (N,N,N-trimethylglycine) and destabilizing (urea) osmolytes on the hydration spheres of biomacromolecules in folded forms (trpzip-1 peptide and hen egg white lysozymeâhewl) and unfolded protein models (glycineâGLY and N-methylglycineâNMG) by means of infrared spectroscopy. GLY and NMG were clearly limited as minimal models for unfolded proteins and should be treated with caution. We isolated the spectral share of water changed simultaneously by the biomacromolecule/model molecule and the osmolyte, which allowed us to provide unambiguous experimental arguments for the mechanism of stabilization/destabilization of proteins by osmolytes. In the case of both types of osmolytes, the decisive factor determining the equilibrium folded/unfolded state of protein was the enthalpy effect exerted on the hydration spheres of proteins in both forms. In the case of stabilizing osmolytes, enthalpy was also favored by entropy, as the unfolded state of a protein was more entropically destabilized than the folded state.
Assuntos
Proteínas , Água , Betaína/química , Desnaturação Proteica , Termodinâmica , Ureia/química , Água/químicaRESUMO
Protein fibrillation leads to formation of amyloids-linear aggregates that are hallmarks of many serious diseases, including Alzheimer's and Parkinson's diseases. In this work, we investigate the fibrillation of a short peptide (K-peptide) from the amyloidogenic core of hen egg white lysozyme in the presence of dimethyl sulfoxide or urea. During the studies, a variety of spectroscopic methods were used: fluorescence spectroscopy and the Thioflavin T assay, circular dichroism, Fourier-transform infrared spectroscopy, optical density measurements, dynamic light scattering and intrinsic fluorescence. Additionally, the presence of amyloids was confirmed by atomic force microscopy. The obtained results show that the K-peptide is highly prone to form fibrillar aggregates. The measurements also confirm the weak impact of dimethyl sulfoxide on peptide fibrillation and distinct influence of urea. We believe that the K-peptide has higher amyloidogenic propensity than the whole protein, i.e., hen egg white lysozyme, most likely due to the lack of the first step of amyloidogenesis-partial unfolding of the native structure. Urea influences the second step of K-peptide amyloidogenesis, i.e., folding into amyloids.
Assuntos
Muramidase , Ureia , Amiloide/metabolismo , Animais , Galinhas/metabolismo , Dicroísmo Circular , Dimetil Sulfóxido/farmacologia , Muramidase/química , Peptídeos , Ureia/química , Ureia/farmacologiaRESUMO
Interactions between a solvent and their co-solute molecules in solutions of peptides are crucial for their stability and structure. The K-peptide is a synthetic fragment of a larger hen egg white lysozyme protein that is believed to be able to aggregate into amyloid structures. In this study, a complex experimental and theoretical approach is applied to study systems comprising the peptide, water, and two co-solutes: trimethylamide N-oxide (TMAO) or dimethyl sulfoxide (DMSO). Information about their interactions in solutions and on the stability of the K-peptide was obtained by FTIR spectroscopy and differential scanning microcalorimetry. The IR spectra of various osmolyte-water-model-peptide complexes were simulated with the DFT method (B3LYP/6-311++G(d,p)). The FTIR results indicate that both solutes are neutral for the K-peptide in solution. Both co-solutes affect the peptide to different degrees, as seen in the shape of its amide I band, and have different influences on its thermal stability. DFT calculations helped simplify the experimental data for easier interpretation.
Assuntos
Dimetil Sulfóxido/química , Metilaminas/química , Peptídeos/química , Varredura Diferencial de Calorimetria , Teoria da Densidade Funcional , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
Intermolecular interactions in aqueous solutions are crucial for virtually all processes in living cells. ATR-FTIR spectroscopy is a technique that allows changes caused by many types of such interactions to be registered; however, binary solutions are sometimes difficult to solve in these terms, while ternary solutions are even more difficult. Here, we present a method of data pretreatment that facilitates the use of the Parallel Factor Analysis (PARAFAC) decomposition of ternary solution spectra into parts that are easier to analyze. Systems of the NMA-water-osmolyte-type were used to test the method and to elucidate information on the interactions between N-Methylacetamide (NMA, a simple peptide model) with stabilizing (trimethylamine N-oxide, glycine, glycine betaine) and destabilizing osmolytes (n-butylurea and tetramethylurea). Systems that contain stabilizers change their vibrational structure to a lesser extent than those with denaturants. Changes in the latter are strong and can be related to the formation of direct NMA-destabilizer interactions.
Assuntos
Acetamidas/química , Espectroscopia de Infravermelho com Transformada de Fourier , Análise FatorialRESUMO
The biology and chemistry of proteins and peptides are inextricably linked with water as the solvent. The reason for the high stability of some proteins or uncontrolled aggregation of others may be hidden in the properties of their hydration water. In this study, we investigated the effect of stabilizing osmolyte-TMAO (trimethylamine N-oxide) and destabilizing osmolyte-urea on hydration shells of two short peptides, NAGMA (N-acetyl-glycine-methylamide) and diglycine, by means of FTIR spectroscopy and molecular dynamics simulations. We isolated the spectroscopic share of water molecules that are simultaneously under the influence of peptide and osmolyte and determined the structural and energetic properties of these water molecules. Our experimental and computational results revealed that the changes in the structure of water around peptides, caused by the presence of stabilizing or destabilizing osmolyte, are significantly different for both NAGMA and diglycine. The main factor determining the influence of osmolytes on peptides is the structural-energetic similarity of their hydration spheres. We showed that the chosen peptides can serve as models for various fragments of the protein surface: NAGMA for the protein backbone and diglycine for the protein surface with polar side chains.
Assuntos
Peptídeos/química , Água/química , Fenômenos Químicos , Glicina/análogos & derivados , Glicina/química , Glicilglicina/química , Metilaminas/química , Simulação de Dinâmica Molecular , Pressão Osmótica , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Ureia/químicaRESUMO
In this study, we established a dynamic micromodel of urinary tract infection to analyze the impact of UT-segment-specific urinary outflow on the persistence of E. coli colonization. We found that the adherence of Dr+ E. coli to bladder T24 transitional cells and type IV collagen is maximal at lowest shear stress and is reduced by any increase in flow velocity. The analyzed adherence was effective in the whole spectrum of physiological shear stress and was almost irreversible over the entire range of generated shear force. Once Dr+ E. coli bound to host cells or collagen, they did not detach even in the presence of elevated shear stress or of chloramphenicol, a competitive inhibitor of binding. Investigating the role of epithelial surface architecture, we showed that the presence of budding cells-a model microarchitectural obstacle-promotes colonization of the urinary tract by E. coli. We report a previously undescribed phenomenon of epithelial cell "rolling-shedding" colonization, in which the detached epithelial cells reattach to the underlying cell line through a layer of adherent Dr+ E. coli. This rolling-shedding colonization progressed continuously due to "refilling" induced by the flow-perturbing obstacle. The shear stress of fluid containing free-floating bacteria fueled the rolling, while providing an uninterrupted supply of new bacteria to be trapped by the rolling cell. The progressive rolling allows for transfer of briefly attached bacteria onto the underlying monolayer in a repeating cascading event.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/química , Escherichia coli/fisiologia , Infecções Urinárias/microbiologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana , Escherichia coli/genética , Humanos , Estresse MecânicoRESUMO
FTIR spectroscopy is one of the best techniques to study intermolecular interactions. However, such an application requires high quality spectra with as little noise as possible, which are often difficult to obtain. One of the main sources of unwanted interference is water vapor. Here a robust method is proposed for automatic, fast and reliable vapor correction of FTIR spectra. The presented least squares approach of vapor subtraction using many vapor spectra and a special residual function provides a much better correction. It does not rely on the researcher's experience, no coefficients are arbitrarily chosen or tweaked, thus such results are more trustworthy and accurate.
RESUMO
Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by â¼50 kJ mol-1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from â¼10-17 s-1 to 10-7 s-1 Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae.
Assuntos
Adesinas Bacterianas/metabolismo , Cistina/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Escherichia coli Uropatogênica/fisiologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aderência Bacteriana , Linhagem Celular Tumoral , Sequência Conservada , Cisteína/química , Transferência de Energia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica , Dobramento de Proteína , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Infrared (IR) spectroscopy is a widely used and invaluable tool in the studies of solvation phenomena in electrolyte solutions. Using state-of-the-art chemometric analysis of a spectral series measured in a concentration-dependent manner, the spectrum of the solute-affected solvent can be extracted, providing a detailed view of the structural and energetic states of the solvent molecules influenced by the solute. Concurrently, ab initio molecular dynamics (AIMD) simulations provide the solvation shell picture at an atomistic detail level and allow for a consistent decomposition of the theoretical IR spectrum in terms of distance-dependent contributions of the solvent molecules. Here, we show for the first time how the chemometric techniques designed with the analysis of experimental data in mind can be harnessed to extract corresponding information from the computed IR spectra for mutual benefit, but without any mutual input. The wide applicability of this two-track approach is demonstrated using lithium bromide solvation in γ-butyrolactone (GBL) as a showcase. GBL is a cyclic ester with extensive applications as a solvent in electrochemistry and we are particularly motivated by its usefulness in the rechargeable cell industry which justifies further studies of lithium cation solvation in GBL. The combination of experiment and simulations firmly asserts the strong solvent structuring character of Li+ and a comparatively weak influence exerted on the solvent by Br-.
RESUMO
The stability of proteins in an aqueous solution can be modified by the presence of osmolytes. The hydration sphere of stabilizing osmolytes is strikingly similar to the enhanced hydration sphere of a protein. This similarity leads to an increase in the protein stability. Moreover, the hydration sphere of destabilizing osmolytes is significantly different. These solutes generate in their surroundings so-called "structurally different water". The addition of such osmolytes causes "dissolution" of the specific protein hydration sphere and destabilizes its folded form. No relationship is seen between the stabilizing/destabilizing properties of osmolytes and their structure-making/-breaking influence on water. Furthermore, their accumulation at the protein surface or their exclusion does not determine the osmolytes' effect on protein stability. An explanation to the osmolytes' stabilizing/destabilizing influence originates in the similarity of water properties in osmolytes and protein solutions. The spectral infrared characteristic of water in an osmolyte solution allowed us to develop practical criteria for classifying solutes as stabilizing or destabilizing agents.
Assuntos
Muramidase/química , Muramidase/metabolismo , Concentração Osmolar , Estabilidade Proteica , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
Proteins' thermal stabilization is a significant problem in various biomedical, biotechnological, and technological applications. We investigated thermal stability of hen egg white lysozyme in aqueous solutions of the following stabilizing osmolytes: Glycine (GLY), N-methylglycine (NMG), N,N-dimethylglycine (DMG), N,N,N-trimethylglycine (TMG), and trimethyl-N-oxide (TMAO). Results of CD-UV spectroscopic investigation were compared with FTIR hydration studies' results. Selected osmolytes increased lysozyme's thermal stability in the following order: Gly>NMG>TMAO≈DMG>TMG. Theoretical calculations (DFT) showed clearly that osmolytes' amino group protons and water molecules interacting with them played a distinctive role in protein thermal stabilization. The results brought us a step closer to the exact mechanism of protein stabilization by osmolytes.
Assuntos
Muramidase/química , Dicroísmo Circular , Estabilidade Enzimática , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Água/químicaRESUMO
Results concerning the thermostability of hen egg white lysozyme in aqueous solutions with stabilizing osmolytes, trimethylamine-N-oxide (TMAO), glycine (Gly), and its N-methyl derivatives, N-methylglycine (NMG), N,N-dimethylglycine (DMG), and N,N,N-trimethylglycine (betaine, TMG), have been presented. The combination of spectroscopic (IR) and calorimetric (DSC) data allowed us to establish a link between osmolytes' influence on water structure and their ability to thermally stabilize protein molecule. Structural and energetic characteristics of stabilizing osmolytes' and lysozyme's hydration water appear to be very similar. The osmolytes increase lysozyme stabilization in the order bulk water < TMAO < TMG < Gly < DMG < NMG, which is consistent with the order corresponding to the value of the most probable oxygen-oxygen distance of water molecules affected by osmolytes in their surrounding. Obtained results verified the hypothesis concerning the role of water molecules in protein stabilization, explained the osmophobic effect, and finally helped to bring us nearer to the exact mechanism of protein stabilization by osmolytes.
Assuntos
Aminas/química , Aminoácidos/química , Proteínas do Ovo/química , Muramidase/química , Água/química , Animais , Betaína/química , Calorimetria , Galinhas , Feminino , Glicina/química , Metilaminas/química , Oxigênio/química , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Sarcosina/química , Soluções/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura de TransiçãoRESUMO
Urinary tract infections caused by Escherichia coli are very common health problem in the developed countries. The virulence of the uropathogenic E. coli Dr(+) IH11128 is determined by Dr fimbriae, which are homopolymeric structures composed of DraE subunits with the DraD protein capping the fiber. In this study, we have analyzed the structural and biochemical properties of biofilms developed by E. coli strains expressing Dr fimbriae with or without the DraD tip subunit and the surface-exposed DraD protein. We have also demonstrated that these E. coli strains form biofilms on an abiotic surface in a nutrient-dependent fashion. We present evidence that Dr fimbriae are necessary during the first stage of bacterial interaction with the abiotic surface. In addition, we reveal that the DraD alone is also sufficient for the initial surface attachment at an even higher level than Dr fimbriae and that chloramphenicol is able to reduce the normal attachment of the analyzed E. coli. The action of chloramphenicol also shows that protein synthesis is required for the early events of biofilm formation. Additionally, we have identified reduced exopolysaccharide coverage in E. coli that express only Dr fimbrial polyadhesins at the cell surface with or without the DraD capping subunit.
Assuntos
Biofilmes , Infecções por Escherichia coli/microbiologia , Escherichia coli Uropatogênica/fisiologia , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Escherichia coli Uropatogênica/genéticaRESUMO
In this paper we present a chemometric method of analysis leading to isolation of Fourier transform infrared (FT-IR) spectra of biomacromolecules (HEW lysozyme, ctDNA) affected by osmolytes (trimethylamine-N-oxide and N,N,N-trimethylglycine, respectively) in aqueous solutions. The method is based on the difference spectra method primarily used to characterize the structure of solvent affected by solute. The cyclical usage of factor analysis allows precise information to be obtained on the shape of "affected spectra" of analyzed biomacromolecules. "Affected spectra" of selected biomacromolecules give valuable information on their structure in the presence of the osmolytes in solution, as well as on the level of perturbation in dependence of osmolyte concentration. The method also gives a possibility of insight into the mechanism of interaction in presented types of systems. It can be easily adapted to various chemical and biochemical problems where vibrational or ultraviolet-visible (UV-Vis) spectroscopy is used.
Assuntos
DNA/isolamento & purificação , Muramidase/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Animais , Bovinos , Galinhas , DNA/análise , DNA/química , Glicina/análogos & derivados , Glicina/química , Metilaminas/química , Muramidase/análise , Muramidase/químicaRESUMO
In this paper, the hydration of a model protein--hen egg white lysozyme in aqueous solution has been presented. The leading method used was FTIR spectroscopy with an application of a technique of semi-heavy water (HDO) isotope dilution. Analysis of spectra of HDO isotopically diluted in water solution of lysozyme allowed us to isolate HDO spectra affected by lysozyme, and thus to characterise the energetic state of water molecules and their arrangement around protein molecules. The number of water molecules and the shape of the affected HDO spectrum were obtained using a classical and a chemometric method. This shape showed that the HDO spectrum affected by lysozyme may be presented as a superposition of two spectra corresponding to HDO affected by N-methylacetamide and the carboxylate anion (of the formic acid). Moreover, based on the difference in intermolecular distances distribution of water molecules (obtained from spectral data), we demonstrated that the lysozyme molecule causes a decrease in population of weak hydrogen bonds, and concurrently increases the probability of an occurrence of short hydrogen bonds in water affected by lysozyme. This conclusion was also confirmed by the molecular dynamics (MD) simulation.
Assuntos
Óxido de Deutério/química , Simulação de Dinâmica Molecular , Muramidase/química , Água/química , Ligação de Hidrogênio , Modelos Moleculares , Muramidase/metabolismo , Soluções , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
BACKGROUND: DraD invasin encoded by the dra operon possesses a classical structure characteristic to fimbrial subunits of the chaperone/usher type. The Ig-fold of the DraD possesses two major characteristics distinguishing it from the family of fimbrial subunits: 1) a distortion of the ß-barrel structure in the region of the acceptor cleft, demonstrated by a disturbance of the main-chain hydrogen bonds network, and 2) an unusually located disulfide bond connecting B and F strands - the localization exclusively observed in the subfamily of DraD/AfaD-type subunits. RESULTS: To evaluate the influence of the DraD-sc specific structural features on its stability and mechanism of thermal denaturation, a series of DSC and FT-IR denaturation experiments were performed giving following conclusions. 1) The DraD-sc is characterized by a low stability (standard Gibbs free energy and enthalpy of unfolding of 18.4 ±1.4 kJ mol(-1) and 131 ±25 kJ mol(-1), respectively) that contrasts strongly with almost infinite stability of the described previously DraE-sc fimbrial protein. 2) The DraD-sc unfolds thermally according to the two state equilibrium model, in contrast to the irreversible kinetically controlled transition of the DraE-sc. 3) The DraD specific disulfide bond is crucial at the folding stage and has little stability effect in the mature protein. CONCLUSIONS: Data published so far emphasize unique biological properties of the DraD invasin as fimbrial subunit: a chaperone independent folding, an usher independent surface localization and the possibility to exist in two forms: as unbound subunits and as loosely bound at fimbrial tip.Presented calorimetric and FT-IR stability data combined with structural correlations has underlined that the DraD invasin is also characterized by unique physicochemical and structural attributes in the context of its belonging to the family of fimbrial subunits.
Assuntos
Adesinas de Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Fímbrias/química , Chaperonas Moleculares/química , Varredura Diferencial de Calorimetria , Dissulfetos , Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Fimbrial adhesins of pathogenic bacteria are linear protein associates responsible for binding to the specific host cell receptors. They are assembled via the chaperone/usher pathway conserved in Gram-negative bacteria. These adhesive organelles are characterized by the high resistance to dissociation and unfolding caused by temperature or chemical denaturants. The self-complemented (SC) recombinant subunits of adhesive structures make up the minimal model used to analyze stability phenomena of these organelles. The SC subunits are both highly stabilized thermodynamically and kinetically. They are characterized by a standard free energy of unfolding of 70-80 kJ/mol and a rate constant of unfolding of 10(-17) s(-1) (half-life of unfolding of 10(8) years at 25 degrees C). The DraE subunit of Dr fimbriae is characterized by a disulfide bond that joins the beginning of the A1 strand with the end of the B strand. Such localization is unique and differentiates this protein from other proteins of the Ig-like family. Sequence analysis shows that many protein subunits of adhesive structures possess cysteines that may form a potential disulfide bond homologous to that of DraE. In this paper, we investigate the influence of this noncanonical disulfide bond on the stability of DraE-sc by constructing a DraE-sc-DeltaSS mutant protein (Cys/Ala mutant). This construct unfolds thermally at a T(m) of 65.4 degrees C, more than 20 degrees C lower than that of the native DraE-sc protein, and possesses a different unfolding mechanism. The calculated standard free energy of unfolding of DraE-sc-DeltaSS is equal to 30 +/- 5 kJ/mol. This allows us to suggest that the disulfide bond is an important stabilizing feature of many fimbrial subunits.
Assuntos
Adesinas Bacterianas/química , Dissulfetos/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Fímbrias Bacterianas/química , Imunoglobulinas/química , Dobramento de Proteína , Subunidades Proteicas/química , Adesinas Bacterianas/genética , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Sequência Conservada , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Ligação de Hidrogênio , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Desnaturação Proteica , Estabilidade Proteica , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
In this paper production of a cold-active esterase EstA from the Antarctic bacterium Pseudoalteromonas sp. 643A in E. coli expression system was described. The purification and biochemical characteristic of EstA were performed in the presence of urea and then compared with results obtained for the esterase with no addition of urea and isolated from the native source. In both cases the cold-active enzyme displayed similar properties. However, the differences concerning thermal activity were observed. The optimal temperature for recombinant esterase in the presence of urea (1 M) was about 15 degrees C lower in comparison with enzyme isolated from the native source. Furthermore, the EstA was found to be more thermolabile in denaturant conditions. The differences were presumably caused by slightly changed protein structure in the presence of urea. The preservation of activity of EstA dissolved in buffer containing 8 M urea suggests that the protein structure is retained and it does not undergo dramatic changes due to high urea concentration. This thesis was confirmed with FT-IR data.
Assuntos
Temperatura Baixa , Esterases/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Desnaturação Proteica , Pseudoalteromonas/enzimologia , Clonagem Molecular , Esterases/química , Concentração de Íons de Hidrogênio , Pseudoalteromonas/genética , Pseudoalteromonas/metabolismoRESUMO
Dr fimbriae of uropathogenic Eschericha coli strains are an example of surface-located adhesive structures assembled via the chaperone-usher pathway. These structures are crucial for specific attachment of bacteria to host receptors. Dr fimbriae are linear associates of DraE proteins, the structure of which is determined by a donor strand complementation between the consecutive subunits. The biogenesis of these structures is dependent on a function of the specific periplasmic chaperone and outer membrane usher proteins. In a consequence of these structural and assembly properties the potential unfolding of a single subunit in a linear associate would cause a destruction of fimbrial adhesion function. This correlates with the observed high resistance of fimbrial structures for denaturation. In this paper we show that the mechanism of thermal denaturation of DraE-sc protein is well described by an irreversible two-state model which is the reduced form of a Lumry-Eyring protein denaturation model. In theory of this model the observed stability of DraE-sc protein is determined by the high activation barrier for the unfolding stage N-->U. The microcalorimetry experiments permit to determine kinetic parameters of the DraE-sc unfolding process: energy of activation of 463.5 +/- 20.8 kJ.mol(-1) and rate constant of order 10(-17) s(-1). This corresponds to the dissociation/unfolding half-life of Dr fimbriae of 10(8) years at 25 degrees C. The FT-IR experiments show that the high stability of DraE is determined by the cooperative rigid protein core. The presented mechanism of kinetic stability of Dr fimbriae is probably universal to adhesive structures of the chaperone-usher type.