Biomedical subjects

S D'Auria

Publications and source records attributed to S D'Auria.

At least 37 records · Page 2Linked to original sources

PubMed2001

Structural characterization and thermal stability of Notothenia coriiceps metallothionein.

Fish and mammalian metallothioneins (MTs) differ in the amino acid residues placed between their conserved cysteines. We have expressed the MT of an Antarctic fish, Notothenia coriiceps, and characterized it by means of multinuclear NMR spectroscopy. Overall, the architecture of the fish MT is very similar to that of mammalian MTs. However, NMR spectroscopy shows that the dynamic behaviour of the two domains is markedly different. With the aid of absorption and CD spectroscopies, we studied the conformational and electronic features of fish and mouse recombinant Cd-MT and the changes produced in these proteins by heating. When the temperature was increased from 20 to 90 degrees C, the Cd-thiolate chromophore absorbance at 254 nm of mouse MT was not modified up to 60 degrees C, whereas the absorbance of fish MT decreased significantly starting from 30 degrees C. The CD spectra also changed quite considerably with temperature, with a gradual decrease of the positive band at 260 nm that was more pronounced for fish than for mouse MT. The differential effect of temperature on fish and mouse MTs may reflect a different stability of metal-thiolate clusters of the two proteins. Such a conclusion is also corroborated by results showing differences in metal mobility between fish and mouse Zn-MT.

Amino Acid Sequence↗

PubMed2001

Enzyme fluorescence as a sensing tool: new perspectives in biotechnology.

The technology for fluorescence protein-sensing is advancing rapidly owing to the continued introduction of new concepts, new fluorophores, and proteins engineered for sensing-specific analytes. Concerns about the reversibility and selectivity of engineered proteins are being addressed by developing biosensors that are based on the utilisation of coenzyme-depleted enzymes. Such biomolecules do not consume the substrate and can exhibit conformational changes upon the binding of the analyte, which can be easily detected as fluorescence change. In addition, concerns about the stability of biosensors can be overcome by using thermostable enzymes isolated from thermophilic microorganisms. Finally, the development of new techniques such as polarization-based sensing, anisotropy-based sensing and lifetime-based sensing, all of which can be accomplished with light-emitting diodes as the light source, is prompting the design of a new class of specific and stable biosensors, as has occurred with blood glucose measurement. These biosensors represent a valid alternative to the conventional clinical chemistry diagnostics.

Biosensing Techniques↗

PubMed2001

On the effect of sodium dodecyl sulfate on the structure of beta-galactosidase from Escherichia coli. A fluorescence study.

An understanding of the structure-function relationship of proteins under different chemical-physical conditions is of fundamental importance for an understanding of their structure and function in cells. In this paper we report the effects of sodium dodecyl sulfate and temperature on the structure of beta-galactosidase from Escherichia coli, as monitored by fluorescence spectroscopy. The structure of the protein was studied in the temperature range of 10-60 degrees C in the absence and presence of sodium dodecyl sulfate by frequency-domain measurement of the intrinsic fluorescence intensity and anisotropy decays. The time-resolved fluorescence data in the absence of SDS indicated that at 10 degrees C the tryptophanyl emission decays were well described by a three exponential decays model, and that the temperature increase resulted in shortening of the long-lived component with little change in the short- and middle-lived components. The addition of SDS to the protein solution also affected the long-lived component. The effects of the detergent and temperature on the enzyme structure were also investigated by means of quenching experiments and anisotropy decays. The obtained results showed that the presence of SDS confers more flexibility to the protein structure, and suggest a strict relation between enzyme activity and protein flexibility.

Acrylamide↗

PubMed2000

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus.

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.

Archaeoglobus fulgidus↗

PubMed2000

A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor.

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration. Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors. Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.

Animals↗

PubMed2000

A protein biosensor for lactate.

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.

Anilino Naphthalenesulfonates↗

PubMed2000

Pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius as probe to monitor the sodium concentrations in the blood.

We describe the isolation and characterization of a pyruvate kinase from the thermophilic eubacterium Bacillus acidocaldarius. This protein appears to be a tetramer composed of four 55-kDa subunits. The intrinsic tryptophan fluorescence of this protein is quenched by approximately 20% upon binding sodium, which occurs with a dissociation constant near 15 mM. Importantly, the intrinsic fluorescence of this pyruvate kinase does not appear to be affected by potassium, magnesium, and calcium at the concentrations found in whole blood. It appears that this pyruvate kinase can provide the basis for a selective protein sensor for sodium with minimal interference from other cations.

Ammonium Sulfate↗

PubMed2000

The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature.

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.

Archaeoglobus fulgidus↗

PubMed2000

Cloning, overexpression, and properties of a new thermophilic and thermostable esterase with sequence similarity to hormone-sensitive lipase subfamily from the archaeon Archaeoglobus fulgidus.

A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demonstrated to be a B"-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [(3)H]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed.

Amino Acid Sequence↗

PubMed2000

Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein.

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5'-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (> or = 50 degrees C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate approximately 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the alpha-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5'-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to approximately 50 degrees C, and (2) stabilizes the covalent protein cofactor linkage up to approximately 70 degrees C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 degrees C of approximately 25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a approximately 20 degrees C higher value on the temperature scale.

Anions↗

PubMed1999

The fluorescence emission of the apo-glucose oxidase from Aspergillus niger as probe to estimate glucose concentrations.

We developed a new method of glucose sensing using an inactive form of glucose oxidase from Aspergillus niger. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8-Anilino-1-naphthalenesulfonic acid (ANS) was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the K(D) of holo-glucose oxidase. These results suggest that apo-glucose oxidase can be used as a reversible nonconsuming sensor for glucose.

Anilino Naphthalenesulfonates↗

PubMed1999

The beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: enzyme activity and conformational dynamics at temperatures above 100 degrees C.

Enzymes from thermophilic organisms are stable and active at temperatures which rapidly denature mesophilic proteins. However, there is not yet a complete understanding of the structural basis of their thermostability and thermoactivity since for each protein there seems to exist special networks of interactions that make it stable under the desired conditions. Here we have investigated the activity and conformational dynamics above 100 degrees C of the beta-glycosidase isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. This has been made possible using a special stainless steel optical pressure cell which allowed us to perform enzyme assays and fluorescence measurements up to 160 degrees C without boiling the sample. The beta-glycosidase from S. solfataricus showed maximal activity at 125 degrees C. The time-resolved fluorescence studies showed that the intrinsic tryptophanyl fluorescence emission of the protein was represented by a bimodal distribution with Lorential shape and that temperature strongly affected the protein conformational dynamics. Remarkably, the tryptophan emission reveals that the indolic residues remain shielded from the solvent even at 125 degrees C, as shown by shielding from quenching and restricted tryptophan solubility. The relationship between enzyme activity and protein structural dynamics is discussed.

Algorithms↗

PubMed1999

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.

Amino Acid Sequence↗

PubMed1999

beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: structure and activity in the presence of alcohols.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95 degrees C. Understanding the structure-activity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus beta-glycosidase expressed in Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.

1-Butanol↗

PubMed1999

Different effects of microwave energy and conventional heat on the activity of a thermophilic beta-galactosidase from Bacillus acidocaldarius.

The effect of microwave (f = 10.4 GHz) irradiation on a thermostable enzyme was experimentally tested, showing that irreversible inactivation is obtained. Enzymatic solutions (500 microliters, with concentrations between 10-100 micrograms/ml) were exposed at 70 degrees C, at SAR levels of 1.1 and 1.7 W/g for 15, 30, 45, or 60 min, and their activity was compared to that of a sample heated in a water bath at the same temperature. The residual activity of the exposed samples depends on enzyme concentration, microwave power level, and exposure time; activity was reduced to 10% in 10 micrograms/ml solutions treated at 1.7 W/g for 60 min. Microwave effects disappeared at concentrations above 50 micrograms/ml. These results were not found following water bath heating at the same temperature and durations.

Bacillus↗

PubMed1998

Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli.

Guanidine-induced denaturation of Sulfolobus solfataricus beta-glycosidase expressed in Escherichia coli, Sbetagly, was investigated at pH 6.5 and 25 degreesC by means of circular dichroism and fluorescence measurements. The process proved reversible when the protein concentration was lower than 0.01 mg mL-1. Moreover, the transition curves determined by fluorescence did not coincide with those determined by circular dichroism, and the GuHCl concentration corresponding at half-completion of the transition increased on raising the protein concentration in the range 0.001-0.1 mg mL-1. Gel filtration chromatography experiments showed that, in the range 2-4 M GuHCl, there was an equilibrium among tetrameric, dimeric, and monomeric species. These findings, unequivocally, indicated that the guanidine-induced denaturation of Sbetagly was not a two-state transition with concomitant unfolding and dissociation of the four subunits. A mechanism involving a dimeric intermediate species was proposed and was able to fit the experimental fluorescence intensity transition profiles, allowing the estimation of the total denaturation Gibbs energy change at 25 degreesC and pH 6.5. This figure, when normalized for the number of residues, showed that, at room temperature, Sbetagly has a stability similar to that of mesophilic proteins.

Chromatography, Gel↗

PubMed1998

Structural analysis of ASCUT-1, a protein component of the cuticle of the parasitic nematode Ascaris lumbricoides.

CUT-1 from the intestinal parasitic nematode Ascaris lumbricoides is a protein component of the insoluble residue of the cuticle, cuticlin. It contains the CUT-1-like domain which is shared by members of a novel family of components of extracellular matrices. The structure and the thermal stability of recombinant CUT-1 from A. lumbricoides (ASCUT-1) were investigated by Fourier-transform infrared (FT-IR) and CD spectroscopy. The data revealed that the secondary structure of the protein at 20 degrees C, both as insoluble inclusion bodies or in soluble form, contains about 50% beta structure, 14% alpha-helix and 25% turns. A tendency of A. lumbricoides CUT-1 to form aggregates was documented by FT-IR spectroscopy which showed also that the addition of SDS disrupts these interactions. Near-ultraviolet CD spectra confirmed these data and suggested that phenylalanine residues are probably involved in intermolecular hydrophobic interactions responsible for the tendency of the protein to aggregate. Near-ultraviolet spectra showed also that part of the cysteine residues forms disulphide bridges responsible for the tertiary architecture of the protein. Finally, FT-IR and CD data revealed that ASCUT-1 is very stable at high temperatures. This stability and the tendency of ASCUT-1 to form aggregates suggest that these properties may be important for a protein which is a component of a particularly resistant extracellular matrix such as the nematode cuticle.

Amino Acid Sequence↗

PubMed1998

Structure-function studies on beta-glycosidase from Sulfolobus solfataricus. Molecular bases of thermostability.

beta-Glycosidase from the extreme thermophilic archaeon Sulfolobus solfataricus is a thermostable tetrameric protein with a molecular mass of 240 kDa which is stable in the presence of detergents and has a maximal activity above 95 degrees C. An understanding of the structure-function relationship of the enzyme under different chemical-physical conditions is of fundamental importance for both theoretical and application purposes. In this paper we report the effect of basic pH values on the structural stability of this enzyme. The structure of the enzyme was studied at pH 10 and in the temperature range 25-97.5 degrees C using circular dichroism, Fourier-transform infrared and fluorescence spectroscopy. The spectroscopic data indicated that the enzyme stability was strongly affected by pH 10 suggesting that the destabilization of the protein structure is correlated with the perturbation of ionic interactions present in the native protein at neutral pHs. These experiments give support to the observation derived from the 3D-structure, that large ion pair networks on the surface stabilize Sulfolobus solfataricus beta-glycosidase.

Circular Dichroism↗