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1.
Biophys J ; 96(3): 997-1007, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19186137

RESUMEN

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D2O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D2O-ESEEM intensities decrease from TOAC(1) to TOAC(8) and TOAC(16) but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC(16) is located in the bilayer leaflet opposite to that of TOAC(1) and TOAC(8). Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D2O-ESEEM intensity is greatest for TOAC(8), indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.


Asunto(s)
Alameticina/química , Alameticina/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Óxidos N-Cíclicos/química , Óxido de Deuterio/metabolismo , Marcadores de Spin , Secuencia de Aminoácidos , Difusión , Espectroscopía de Resonancia por Spin del Electrón , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Datos de Secuencia Molecular , Fosfolípidos/química , Fosfolípidos/metabolismo , Temperatura
2.
Biochemistry ; 48(35): 8343-54, 2009 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-19642639

RESUMEN

Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labeled on class I -SH groups in the presence of glycerol, or on class II -SH groups in the absence of glycerol. The class I-labeled preparations retain full enzymatic activity, whereas the class II-labeled preparations are at least partially inactivated. This provides an excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) can provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labeled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. 2H-ESEEM spectra of membranes dispersed in D2O reveal that class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or class II groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures (<150 K), but a more homogeneous conformational state at higher temperatures that is characterized by a single phase-memory T2M relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching an almost pure Lorentzian line shape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.


Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Sulfuro de Hidrógeno/química , ATPasa Intercambiadora de Sodio-Potasio/química , Marcadores de Spin , Animales , Cristalización , Electrones , Conformación Molecular , Tiburones , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos , Temperatura
3.
Biophys Chem ; 94(1-2): 107-20, 2001 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-11744195

RESUMEN

Two molecular dynamics simulations have been performed for 2 ns, at room temperature, on fully hydrated wild type and Cys3Ala/Cys26Ala double-mutant azurin, to investigate the role of the unique disulfide bridge on the structure and dynamics of the protein. The results show that the removal of the [bond]SS[bond] bond does not affect the structural features of the protein, whereas alterations of the dynamical properties are observed. The root mean square fluctuations of the atomic positions are, on average, considerably reduced in the azurin mutant with respect to the wild type form. The number of intramolecular hydrogen bonds between protein backbone atoms that are lost during the simulation, with respect to the starting configuration, are reduced in the absence of the disulfide bond. The analysis of the dynamical cross-correlation map, characterising the protein co-ordinated internal motions, demonstrates in the mutated azurin a significant decrease in anti-correlated displacements between protein residues, with the only exception occurring in the region of the mutation sites. The overall findings show a relevant reduction in flexibility as a consequence of the disulfide bridge depletion in azurin, suggesting that the [bond]SS[bond] bond is a structural element which significantly contributes to the dynamic properties of the native protein.


Asunto(s)
Azurina/química , Disulfuros/química , Modelos Moleculares , Biología Computacional , Enlace de Hidrógeno , Conformación Proteica
4.
Biophys Chem ; 82(1): 9-22, 1999 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-17030337

RESUMEN

The effect of heavy water on the structure and dynamics of copper plastocyanin as well as on some aspects of the solvent dynamics at the protein-solvent interfacial region have been investigated by molecular dynamics simulation. The simulated system has been analyzed in terms of the atomic root mean square deviation and fluctuations, intraprotein H-bond pattern, dynamical cross-correlation map and the results have been compared with those previously obtained for plastocyanin in H2O (Ciocchetti et al. Biophys. Chem. 69 (1997), 185-198). The simulated plastocyanin structure in the two solvents, averaging 1 ns, is very similar along the beta-structure regions, while the most significant differences are registered, analogous to the turns and the regions likely involved in the electron transfer pathway. Moreover, plastocyanin in D2O shows an increase in the number of both the intraprotein H-bonds and the residues involved in correlated motions. An analysis of the protein-solvent coupling evidenced that D2O makes the H-bond formation more difficult with the solvent molecules for positively charged and polar residues, while an opposite trend is observed for negatively charged residues. On the other hand, the frequency of exchange of the solvent molecules involved in the protein-solvent H-bond formation is significantly depressed in D2O. The results are discussed also in connection with protein functionality and briefly with some experimental results connected with the thermostability of proteins in D2O.

5.
Biophys Chem ; 63(2-3): 211-9, 1997 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-17029824

RESUMEN

The effects of cooling rate and of solvent properties on the active site heterogeneity of two copper proteins, azurin and plastocyanin, have been investigated at low temperature by electron paramagnetic resonance spectroscopy. The spectra of theses proteins have been analyzed, by an accurate computer simulation, in terms of a distribution of some relevant spin-Hamiltonian parameters. The results show that the structural heterogeneity of both proteins, quantified by the width of the distribution in the g and A tensors, is affected by both the freezing procedure and the solvent composition. In particular, the g distribution width is found to be reduced in the slow cooling regime; such a reduction appearing more significant when glycerol is added to the protein solutions. Despite of the similarity in the copper ion microenvironments of the two proteins, the effects are more pronounced in azurin. The results are discussed also in connection with the role played by the solvent and the rate of freezing in featuring the conformational substate landscape.

6.
J Inorg Biochem ; 91(3): 463-9, 2002 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-12175938

RESUMEN

The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric <--> g and <--> A tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins. Such a reduction, quantified by the standard deviations sigma(g axially) and sigma(A axially) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(g axially) and sigma(A axially) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N(-)(3) and SCN(-) addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions.


Asunto(s)
Aniones , Azurina/química , Proteínas Bacterianas/química , Conformación Proteica , Sustitución de Aminoácidos , Azurina/genética , Azurina/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Metaloproteínas/química , Metaloproteínas/metabolismo , Mutagénesis Sitio-Dirigida , Distribución Normal , Paracoccus/genética , Paracoccus/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
7.
Int J Biol Macromol ; 31(4-5): 163-70, 2003 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-12568924

RESUMEN

The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry-Eyring model, N if U-->F, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 degrees C, while the unfolding free energy, deltaG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed DeltaG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E(a), as derived from the kinetic stability analysis.


Asunto(s)
Azurina/química , Cobre/química , Zinc/química , Alanina/química , Rastreo Diferencial de Calorimetría , Cisteína/química , Cinética , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , Temperatura , Termodinámica
8.
Artículo en Inglés | MEDLINE | ID: mdl-8073842

RESUMEN

We studied 179 psoriatic patients by semistructured colloquia and psychometric tests and determined their cutaneous psycho-neurophysiological profiles by biofeedback methods. The Paykel scale for stressful events showed that 72% of psoriatics had experienced significant stressful events about one month before the appearance of the psoriasis. The Zung test for anxiety and depression showed a high level of anxiety in the psoriatic patients. 64% of the patients who were treated by BFBtraining had a decrease in their PASI index for severity and the extent of the disease and also fewer recurrences at the one-year follow-up. The results of the World Experience Inventory indicated difficulties related to body image and to relationships with others. Psoriasis influenced the sexuality of the patients. It is always difficult when one is afflicted by ill health to enjoy life and the general scores of SWL (Satisfaction with Life), were significantly lower than those of a control group.


Asunto(s)
Psoriasis/psicología , Adulto , Anciano , Ansiedad/complicaciones , Depresión/diagnóstico , Humanos , Persona de Mediana Edad , Psoriasis/patología , Pruebas Psicológicas , Calidad de Vida , Estrés Psicológico/complicaciones
10.
Eur Biophys J ; 37(8): 1351-60, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18560824

RESUMEN

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of beta-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations 0.13 mM an exothermic peak also appears, above 90 degrees C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu(2+) or Zn(2+) ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10 degrees C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.


Asunto(s)
Cobre/farmacología , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Temperatura , Zinc/farmacología , Amiloide/metabolismo , Benzotiazoles , Rastreo Diferencial de Calorimetría , Cationes Bivalentes/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Nefelometría y Turbidimetría , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Tiazoles/metabolismo , Triptófano/metabolismo
11.
Orig Life Evol Biosph ; 36(5-6): 597-603, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17136430

RESUMEN

Absorption or emission spectroscopy is a powerful tool for detecting chemical compounds, diluted in fluid media: the sensitivity of this technique depends on the optical path of the source radiation, on the spectral window used for analysis and on the spectrometer performances. In this view, we designed and produced the first prototypes of an integrated scanning Fourier Transform Microinterferometer with Mach-Zehnder geometry, by using MEOS (Micro Electro Optical Systems) technologies. The microdevice, obtained by fabricating integrated optical waveguides on LiNbO(3) (LN) crystals, is electrically driven, without moving parts, by exploiting the electrooptical properties of the material. The microdevice operates the Fourier Transform of the input radiation spectral distribution, which can be reconstructed starting from the output signal by means of Fast Fourier Transform (FFT) techniques. The microinterferometer weights few grams, the power consumption is of a few mW and, in principle, can operate in the LN transmittance range (0.36-4.5 microm). The microinterferometer performances were preliminary tested in the (0.4-1.7 microm) spectral window. In the Visible region (0.4-0.7 mum) this microsystem demonstrated a spectral resolution suitable for detecting the characteristic lines of the solar spectrum together with the absorption bands of common gases present in Earth's atmosphere. In a further experiment we tested its performances for gas trace detection by using a calibrated NO(2) optical gas cell, showing the possibility to reveal up to 10 ppb, when suitable optical paths are used. Finally, colorimetry tests for the titration of an organic dye (E131) in alcohol solution are presented.


Asunto(s)
Interferometría/instrumentación , Niobio/química , Óxidos/química , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Cristalización , Diseño de Equipo , Gases/análisis , Gases/química , Interferometría/métodos , Miniaturización , Dióxido de Nitrógeno/química , Reproducibilidad de los Resultados , Espectroscopía Infrarroja por Transformada de Fourier/métodos
12.
Appl Opt ; 40(33): 6062-71, 2001 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-18364904

RESUMEN

The theory of the multiple-pass cell based on the use of retroreflectors is presented. As a result of this study, it is shown that it is possible to construct an enhanced White cell with zero geometric loss. Starting from theoretical considerations of the design of a new monolithic multiple-face retroreflector, a multiple-pass cell is proposed. Ray-tracing simulations indicate that this cell is easy to align and has zero geometric loss over a very long optical path.

13.
Appl Opt ; 32(18): 3257-65, 1993 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-20829942

RESUMEN

We discuss the problem arising from the nonuniform distribution of spectral transmittance of the instrument's shielding dome. Recently, by using high-resolution atmospheric radiation software, itbecame possible to evaluate both the real downward IR spectral flux density and the transmitted spectral flux density of the instrument's dome. By using a simplified model we obtained a theoretical formula that describes the effect of dome transmittance on the instrument's response. By the formula, the effective dome transmittance (average dome transmittance weighted by the spectral flux density) is directly proportional to the calibration constant of the instrument. In order to obtain a quantitative relationship we computed the effective dome transmittance for several domes and model atmospheres. ccording to our results the maximum difference in effective dome transmittance of individual domes is 20% for Eppley-type silicon domes and 10% for polyethylene domes. These relatively large differencesmust be corrected when the domes are replaced. The effective dome transmittance shows strongcorrelation with precipitable water and the total downward IR flux density. The combined effect on thecalibration factor is a maximum 2% for the Eppley domes and 5% for the polyethylene domes. By usingthe linear regression method these types of error can be minimized.

14.
Appl Opt ; 21(9): 1578-87, 1982 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-20389899

RESUMEN

A modification of the Twomey-Phillips linear constrained inversion method is used to retrieve aerosol size spectra from simulated spectral optical depths in the wavelength range from 0.37 to 2.2 microm. It is found that the transition between nonphysical solutions to well-behaved ones, which is driven by the smoothing parameter gamma is gradual and that negative components, when present in the solution, always belong to the smallest particle range. When values of gamma greater than 0.05 are required to obtain well-behaved solutions, a strong bias on the retrieved solution is posed by the form of the first-guess solution. Moreover, even if the retrieved size distribution computed for low values of gamma is not well-behaved, much information on the shape of the true solution in the medium and large particle range is contained in the retrieved solution. The effect of realistic random errors added to the simulated optical depths is also discussed.

15.
Appl Opt ; 26(15): 3043-51, 1987 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-20490007

RESUMEN

A model for the atmospheric correction of data remotely sensed over the sea at different altitudes is described in detail and validated through experimental data measured in the North Adriatic Sea during the Adria 83 campaign by a modular multispectral scanner.

16.
Appl Opt ; 24(17): 2859-64, 1985 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-18223968

RESUMEN

A spectroradiometer was developed for use on the ground and aboard ship. The instrument separates global and diffuse solar radiation with a rotating shading band. Calibration results are compared with those of other sun photometers. The instrument was also mounted on a stabilized platform during an experimental trial aboard ship on the Tyrrhenian Sea. Results are comparable with those obtained by handheld sun photometer.

17.
Appl Opt ; 36(6): 1328-36, 1997 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-18250807

RESUMEN

A linear relationship between the variations in radiance measured in the visible and near infrared by satellites and variations of aerosol-extinction profile has been derived, hence reducing the problem to that of solving a Fredholm integral equation of the first kind. The retrieved profiles, by the linear constrained-inversion method, have proved to be accurate in the lower atmosphere, even if simultaneous changes are taking place in the stratosphere. Variations in the stratosphere, however, are retrieved with a lesser degree of accuracy and only as long as no variations occur in the tropospheric haze.

18.
Appl Opt ; 36(30): 8031-41, 1997 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-18264333

RESUMEN

A database management system has been realized that, by taking physical and chemical properties (the complex refractive index and the size distribution) of basic components as its starting point, allows the user to obtain optical properties of default as well as user-defined aerosol classes. Default classes are defined in accordance with the most widely known and used aerosol models. We obtain user-defined classes by varying the mixing ratio of components, creating new mixtures of default components, or by defining user components, thereby supplying the size distribution and the refractive index. The effect of relative humidity (RH) on the refractive index and the size distribution is properly accounted for up to RH = 99%. The two known mechanisms of obtaining classes from components are allowed (internal or external mixing).

19.
Appl Opt ; 28(12): 2199-201, 1989 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-20555496

RESUMEN

A method of correcting for a shadowband is presented, whatever the rotation axis orientation; the only proviso is that it lie in the horizontal plane.

20.
Eur Biophys J ; 30(3): 171-8, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11508836

RESUMEN

A comparative investigation of the effects of cooling rate and solvent physicochemical properties on the structural heterogeneity of wild-type and disulfide bond depleted azurin (Cys3Ala/Cys26Ala) and of amicyanin has been performed by EPR spectroscopy and computer simulation. By describing the spectral features of the EPR spectra in terms of Gaussian distributions of the components of the g and A tensors of the spin Hamiltonian, we have shown that either the cooling rate or the solvent composition affect the structural heterogeneity of the proteins. Such a heterogeneity has been quantified by the standard deviations sigmag and sigmaA of the parallel components of the axially symmetric tensors. In particular, both parameters become smaller after the slow cooling cycle; such a reduction is more significant when glycerol is added as cosolvent to the protein solutions. The comparison of the deltag and sigmaA values found, for the copper proteins investigated, highlights that the reduction is more marked in the azurins compared to amicyanin and that the Cys3Ala/Cys26Ala azurin mutant has a structural heterogeneity lower than that shown by the wild-type protein. The remarkable similarity of the copper coordination sphere of the proteins suggests a more rigid structure of the azurin protein matrix in the absence of the disulfide bridge compared to wild-type azurin and of amicyanin with respect to both forms of azurin. The former result establishes an important role for the -SS- bond in modulating the flexibility of wild-type azurin.


Asunto(s)
Azurina/química , Azurina/genética , Proteínas Bacterianas/química , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Alanina/química , Cisteína/química , Metaloproteínas , Modelos Estadísticos , Mutación , Conformación Proteica
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