RESUMEN
The 33-mer gliadin peptide, LQLQPF(PQPQLPY)3PQPQPF, is a highly immunogenic peptide involved in celiac disease and probably in other immunopathologies associated with gliadin. Herein, dynamic light scattering measurements showed that 33-mer, in the micromolar concentration range, forms polydisperse nano- and micrometer range particles in aqueous media. This behaviour is reminiscent of classical association of colloids and we hypothesized that the 33-mer peptide self-assembles into micelles that could be the precursors of 33-mer oligomers in water. Deposition of 33-mer peptide aqueous solution on bare mica generated nano- and microstructures with different morphologies as revealed by atomic force microscopy. At 6 µM, the 33-mer is organised in isolated and clusters of spherical nanostructures. In the 60 to 250 µM concentration range, the spherical oligomers associated mainly in linear and annular arrangements and structures adopting a "sheet" type morphology appeared. At higher concentrations (610 µM), mainly filaments and plaques immersed in a background of nanospherical structures were detected. The occurrence of different morphologies of oligomers and finally the filaments suggests that the unique specific geometry of the 33-mer oligomers has a crucial role in the subsequent condensation and organization of their fractal structures into the final filaments. The self-assembly process on mica is described qualitatively and quantitatively by a fractal diffusion limited aggregation (DLA) behaviour with the fractal dimension in the range of 1.62 ± 0.02 to 1.73 ± 0.03. Secondary structure evaluation of the oligomers by Attenuated Total Reflection FTIR spectroscopy (ATR-FTIR) revealed the existence of a conformational equilibrium of self-assembled structures, from an extended conformation to a more folded parallel beta elongated structures. Altogether, these findings provide structural and morphological information about supramolecular organization of the 33-mer peptide, which might offer new perspectives for the understanding and treatment of gliadin intolerance disorders.
Asunto(s)
Gliadina/química , Multimerización de Proteína , Secuencia de Aminoácidos , Micelas , Datos de Secuencia Molecular , Agregado de ProteínasRESUMEN
Arginine-rich peptides receive increased attention due to their capacity to cross different types of membranes and to transport cargo molecules inside cells. Even though peptide-induced destabilization has been investigated extensively, little is known about the peptide side-chain and backbone orientation with respect to the bilayer that may contribute to a molecular understanding of the peptide-induced membrane perturbations. The main objective of this work is to provide a detailed description of the orientation of arginine peptides in the lipid bilayer of PC and negatively charged PG liposomes using ATR-IR spectroscopy and molecular modeling, and to relate these orientational preferences to lipid bilayer destabilization. Molecular modeling showed that above the transition temperature arginine side-chains are preferentially solvent-directed at the PC/water interface whereas several arginine side-chains are pointing towards the PG hydrophobic core. IR dichroic spectra confirmed the orientation of the arginine side chains perpendicular to the lipid-water interface. IR spectra shows an randomly distributed backbone that seems essential to optimize interactions with the lipid membrane. The observed increase of permeation to a fluorescent dye is related to the peptide induced-formation of gauche bonds in the acyl chains. In the absence of hydrophobic residues, insertion of side-chains that favors phosphate/guanidium interaction is another mechanism of membrane permeabilization that has not been further analyzed so far.
Asunto(s)
Arginina/química , Lípidos de la Membrana/química , Péptidos/química , Péptidos de Penetración Celular/química , Membrana Dobles de Lípidos/química , Conformación Molecular , Simulación de Dinámica Molecular , Transición de Fase , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura de TransiciónRESUMEN
The present study determined alpha-tocopherol mass transfer from an alpha-tocopherol-rich emulsion to LDL and HDL, and assessed the potential of different mechanisms to modulate alpha-tocopherol transfers. Emulsion particles rich in alpha-tocopherol were incubated in vitro with physiological concentrations of LDL or HDL. The influence of plasma proteins was assessed by adding human lipoprotein poor plasma (LPP) fraction with intact vs heat inactivated PLTP, or with a specific cholesteryl ester transfer protein (CETP) inhibitor, or by adding purified PLTP or pig LPP which lacks CETP activity. After 4 h incubation in absence of LPP, alpha-tocopherol content was increased by ~80% in LDL and ~160% in HDL. Addition of LPP markedly enhanced alpha-tocopherol transfer leading to 350-400% enrichment in LDL or HDL at 4 h. Higher (~10 fold) enrichment was achieved after 20 h incubation with LPP. Facilitation of alpha-tocopherol transfer was (i) more than 50% higher with human vs pig LPP (despite similar PLTP phospholipid transfer activity), (ii) reduced by specific CETP activity inhibition, (iii) not fully suppressed by heat inactivation, and (iv) not restored by purified PLTP. In conclusion, alpha-tocopherol content in LDL and HDL can be markedly raised by rapid transfer from an alpha-tocopherol-rich emulsion. Our results indicate that alpha-tocopherol mass transfer between emulsion particles and lipoproteins is mediated by more than one single mechanism and that this transfer may be facilitated not only by PLTP but likely also by other plasma proteins such as CETP.
Asunto(s)
Emulsiones/química , Lípidos/química , Lipoproteínas/química , Vitamina E/química , Vitamina E/metabolismo , Animales , Humanos , Lipoproteínas/sangre , Lipoproteínas/metabolismo , Modelos Biológicos , Estructura Molecular , PorcinosRESUMEN
The inflammatory effect of unmethylated CpG DNA sequences represents a major obstacle to the use of cationic lipids for in vivo gene therapy. Although the mechanism of CpG-induced inflammatory response is rather well understood nowadays, few solutions have been designed to circumvent this effect in gene therapy experiments. Our previous work has shown that a refractory state towards inflammation can be elicited by preinjecting cationic liposomes. Here, we present evidence that diC14-amidine liposomes confer new anti-inflammatory properties to phospholipids from low-density lipoprotein (LDL) and even to synthetic phospholipids for which such an observation has not been reported so far. Whereas oxidation of LDL lipids was a prerequisite for any anti-inflammatory activity, lipid oxidation is no longer required in our experiments, suggesting that cationic lipids transport phospholipids through a different route and affect different pathways. This opens up new possibilities for manipulating inflammatory responses in gene therapy protocols but also in a general manner in immunological experiments.
Asunto(s)
Amidinas/farmacología , Antiinflamatorios no Esteroideos/farmacología , Inflamación/fisiopatología , Fosfolípidos/farmacología , Apoptosis/efectos de los fármacos , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Humanos , Inflamación/genética , LiposomasRESUMEN
LmrP is an electrogenic H(+)/drug antiporter that extrudes a broad spectrum of antibiotics. Five carboxylic residues are implicated in drug binding (Asp142 and Glu327) and proton motive force-mediated restructuring (Asp68, Asp128 and Asp235). ATR-FTIR (Attenuated Total Reflection - Fourier Transform Infrared) and tryptophan quenching experiments revealed that phosphatidylethanolamine (PE) is required to generate the structural intermediates induced by ionization of carboxylic residues. Surprisingly, no ionization-induced conformational changes were detectable in the absence of PE, suggesting either that carboxylic acid residues do not ionize or that ionization does not lead to any conformational change. The mean pKa of carboxylic residues evaluated by ATR-FTIR spectroscopy was 6.5 for LmrP reconstituted in PE liposomes, whereas the pKa calculated in the absence of PE was 4.6. Considering that 16 of the 19 carboxylic residues are located in the extramembrane loops, the pKa values obtained in the absence and in the presence of PE suggest that the interaction of the loop acid residues with the membrane interface depends on the lipid composition.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Fosfatidiletanolaminas/química , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Proteínas Bacterianas/genética , Ácido Glutámico/química , Ácido Glutámico/genética , Proteínas de Transporte de Membrana/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Especificidad por Sustrato , Triptófano/análisisRESUMEN
Two chitinases, able to use tetra-N-acetylglucosamine, chitin and chitosan as substrates, were characterized after purification from Carica papaya latex. The complete amino acid sequence of the major form and about 40% of the minor one were determined through proteolytic digestions and mass spectroscopy analysis. Sequencing demonstrated that both papaya chitinases are members of the family 19 of glycosyl hydrolases (GH19). Based on the known 3-D structures of other members of family GH19, it was expected that papaya chitinases would adopt all-alpha structures. However, circular dichroism and infrared spectroscopy indicated, for the papaya chitinases, a content of 15-20% of extended structures besides the expected 40% of alpha helices. Since the fully sequenced papaya chitinase contains a large number of proline residues the possibility that papaya chitinase contains polyproline II stretches was examined in the context of their resistance against proteolytic degradation.
Asunto(s)
Carica/química , Quitinasas/química , Secuencia de Aminoácidos , Quitinasas/genética , Quitinasas/aislamiento & purificación , Dicroismo Circular , Endopeptidasas/química , Fluorescencia , Hidrolasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Espectrofotometría InfrarrojaRESUMEN
Fourier transform infrared (FT-IR) spectroscopy has become a powerful tool for biodiagnostics and cell line classification. Typical experimental perturbations included in spectra are baseline shift and scale variation between spectra. They have to be removed by data preprocessings to allow further data analysis and classification. In this work, we addressed baseline shift corrections and normalizations in attenuated total reflection (ATR) FT-IR spectra. We compared the efficiency of several preprocessing methods with series of spectra containing typical perturbations (baseline shift, scaling factor, and noise) and a priori known definite spectral difference. Several baseline-correction and normalization possibilities were evaluated. Our results were generally sensitive, selective, and robust with respect to baseline and scaling. Full-range scaling generated more false-positive results. Use of first- and second-derivative spectra was tested. Results obtained on model spectra were confirmed with series of spectra from sensitive and multidrug-resistant leukemia K562 cells. We showed that the use of derived spectra did not provide more efficiency and required additional preprocessing such as smoothing to obtain results similar to those obtained from non-derived ones. On the other hand, results obtained with derivatives were less sensitive to scaling, a useful feature when scaling is problematic.
Asunto(s)
Espectroscopía Infrarroja por Transformada de Fourier/métodos , Antibióticos Antineoplásicos/farmacología , Daunorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Células K562 , Análisis EspectralRESUMEN
LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Transporte de Membrana/fisiología , Acrilamida/farmacología , Ácido Aspártico/química , Proteínas Bacterianas/química , Bencimidazoles/farmacología , Transporte Biológico , Membrana Celular/metabolismo , Citosol/química , Relación Dosis-Respuesta a Droga , Resistencia a Múltiples Medicamentos , Concentración de Iones de Hidrógeno , Lactococcus lactis/metabolismo , Ligandos , Liposomas/metabolismo , Proteínas de Transporte de Membrana/química , Unión Proteica , Estructura Terciaria de Proteína , Proteolípidos/química , Protones , Sefarosa/química , Espectroscopía Infrarroja por Transformada de Fourier , Tetraciclina/química , Factores de Tiempo , Triptófano/químicaRESUMEN
The present research investigates whether infrared spectra can be related to the biological characteristics of glioma cell lines. We used nine human glioma cell lines for which a series of in vitro and in vivo biological features had already been established [Glia 36 (2001) 375] and were able to show that their characteristic infrared spectra reflect their in vitro migration (i.e., motility and invasiveness) properties and their in vivo aggressiveness. More particularly, the infrared data evidenced correlations at the level of the lipid/protein ratio. These relationships were found to be tissue-dependent when controlled on seven pancreatic carcinoma cell lines. We also showed that oligodendroglial and astrocytic tumor cells, whose identification remains difficult, can easily be identified by their infrared spectra in the lipid acyl chain region as well as in the nucleic acid region. We concluded that infrared spectroscopy could usefully complement information provided by more conventional diagnostic and prognostic (e.g., morphological and molecular) approaches.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral/metabolismo , Línea Celular Tumoral/efectos de la radiación , Glioma/metabolismo , Rayos Infrarrojos , Espectrofotometría Infrarroja/métodos , Animales , Astrocitos/citología , Astrocitos/metabolismo , Astrocitos/efectos de la radiación , Neoplasias Encefálicas/clasificación , Neoplasias Encefálicas/diagnóstico , Diferenciación Celular/fisiología , División Celular/fisiología , Línea Celular Tumoral/trasplante , Linaje de la Célula/fisiología , Movimiento Celular/fisiología , Modelos Animales de Enfermedad , Glioma/clasificación , Glioma/diagnóstico , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica/diagnóstico , Trasplante de Neoplasias , Oligodendroglía/citología , Oligodendroglía/metabolismo , Oligodendroglía/efectos de la radiación , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/metabolismo , Valor Predictivo de las Pruebas , Tasa de SupervivenciaRESUMEN
As more and more high-resolution structures of proteins become available, the new challenge is the understanding of these small conformational changes that are responsible for protein activity. Specialized difference Fourier transform infrared (FTIR) techniques allow the recording of side-chain modifications or minute secondary structure changes. Yet, large domain movements remain usually unnoticed. FTIR spectroscopy provides a unique opportunity to record (1)H/(2)H exchange kinetics at the level of the amide proton. This approach is extremely sensitive to tertiary structure changes and yields quantitative data on domain/domain interactions. An experimental setup designed for attenuated total reflection and a specific approach for the analysis of the results is described. The study of one membrane protein, the gastric H(+),K(+)-ATPase, demonstrates the usefulness of (1)H/(2)H exchange kinetics for the understanding of the molecular movement related to the catalytic activity.
Asunto(s)
Membrana Celular/metabolismo , Deuterio , Hidrógeno , Espectrofotometría Infrarroja/métodos , Adenosina Trifosfatasas/química , Adenosina Trifosfato/química , Calorimetría , Catálisis , ATPasa Intercambiadora de Hidrógeno-Potásio/química , Cinética , Modelos Químicos , Modelos Estadísticos , Distribución Normal , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Protones , Espectroscopía Infrarroja por Transformada de Fourier , Estómago/enzimología , Factores de Tiempo , AguaRESUMEN
Membrane fusion proceeds via a merging of two lipid bilayers and a redistribution of aqueous contents and bilayer components. It involves transition states in which the phospholipids are not arranged in bilayers and in which the monolayers are highly curved. Such transition states are energetically unfavourable since biological membranes are submitted to strong repulsive hydration electrostatic and steric barriers. Viral membrane proteins can help to overcome these barriers. Viral proteins involved in membrane fusion are membrane associated and the presence of lipids restricts drastically the potential of methods (RMN, X-ray crystallography) that have been used successfully to determine the tertiary structure of soluble proteins. We describe here how IR spectroscopy allows to solve some of the problems related to the lipid environment. The principles of the method, the experimental setup and the preparation of the samples are briefly described. A few examples illustrate how attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy can be used to gain information on the orientation and the accessibility to the water phase of the fusogenic domain of viral proteins. Recent developments suggest that the method could also be used to detect changes located in the membrane domains and to identify intermediate structural states involved in the fusion process.
Asunto(s)
Espectroscopía Infrarroja por Transformada de Fourier/métodos , Proteínas Virales de Fusión/química , Amidas/química , Deuterio/química , Hidrógeno/química , Intercambio Iónico , Cinética , Fusión de Membrana , Estructura Terciaria de ProteínaRESUMEN
The biochemical composition and biophysical behaviour of pulmonary surfactant samples isolated from healthy Belgian White and Blue (BWB) and Holstein Friesian (HF) calves have been investigated and compared. Interesting differences in composition have been demonstrated. In particular, a higher level of total hydrophobic surfactant-associated proteins (SP) (due to higher levels of SP-B and SP-C) is reported in HF calves compared to BWB calves. Higher levels of phosphatidylcholine (PC) and especially the disaturated form of PC were also found in HF as compared to BWB calves. No immediate effect on the surface tension properties evaluated by the pulsating bubble surfactometer was found between the surfactant samples of the two breeds under physiological conditions. However, since a high content of disaturated PC and the presence of the SP-B and SP-C are thought to be essential for the surface activity, we propose that the reported modifications could contribute to the apparently lower resistance of the BWB calves to respiratory troubles in comparison with HF calves.
Asunto(s)
Bovinos , Pulmón/química , Surfactantes Pulmonares/química , Animales , Líquido del Lavado Bronquioalveolar/química , Femenino , Salud , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Fosfolípidos/análisis , Proteínas Asociadas a Surfactante Pulmonar/análisis , Tensión SuperficialRESUMEN
During the last few years, Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) has become one of the most powerful methods to determine the structure of biological materials and in particular of components of biological membranes, like proteins which cannot be studied by X-ray crystallography and NMR. Indeed, ATR-FTIR method requires little amount of material, gives valuable information about the secondary structure, orientation and tertiary structure changes in peptides and proteins. Moreover, this technique can be used in the presence of lipids and hence provides an excellent tool to study membrane proteins in their natural environment. In this review, we describe how structural information about the catalytic cycle of membrane proteins can be gained by combining ATR-FTIR spectroscopy and mutagenesis. In particular, results obtained about the structure and function of the nucleotide binding domains (NBD) of P-glycoprotein (Pgp), a multidrug transporter involved in cancer cells resistance to chemotherapy, are described.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Catálisis , Cristalografía por Rayos X , Mutagénesis , Resonancia Magnética Nuclear Biomolecular , Mutación Puntual , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-ActividadRESUMEN
Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+-ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane-anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N-terminal region, a M1-M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2-K conformations, but the peptide generated in the presence of vanadate begins 12 amino-acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate-induced conformation. In the M5-M6 region, the membrane-anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7-M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.
Asunto(s)
ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Estómago/enzimología , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Membrana Celular/enzimología , Electroforesis en Gel de Poliacrilamida , ATPasa Intercambiadora de Hidrógeno-Potásio/química , Ligandos , Unión Proteica , Conformación Proteica , Porcinos , Tripsina/metabolismo , Vanadatos/química , Vanadatos/metabolismoRESUMEN
Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented alpha-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of (1)H/(2)H exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Hidrógeno , Hidrólisis , Relación Estructura-ActividadRESUMEN
We have studied a group of fusion peptides of influenza hemagglutinin in which the N-terminal amino acid, Gly (found in the wild-type peptide), has been systematically substituted with Ala, Ser, Val, or Glu. The activity of the intact hemagglutinin protein with these same substitutions has already been reported. As a measure of the extent of modulation of intrinsic membrane curvature by these peptides, we determined their effects on the polymorphic phase transition of dipalmitoleoylphosphatidylethanolamine. The wild-type peptide is the only one that, at pH 5, can substantially decrease the temperature of this transition. This is also the only form in which the intact protein promotes contents mixing in cells. The Ala and Ser mutant hemagglutinins exhibit a hemifusion phenotype, and their fusion peptides have little effect on lipid polymorphism at low pH. The two mutant proteins that are completely fusion inactive are the Val and Glu mutant hemagglutinins. The fusion peptides from these forms significantly increase the polymorphic phase transition temperature at low pH. We find that the effect of the fusion peptides on membrane curvature, as monitored by a shift in the temperature of this polymorphic phase transition, correlates better with the fusogenic activities of the corresponding protein than do measurements of the isotropic (31)P NMR signals or the ability to induce the fusion of liposomes. The inactivity of the hemagglutinin protein with the hydrophobic Val mutation can be explained by the change in the angle of membrane insertion of the helical fusion peptide as measured by polarized FTIR. Thus, the nature of the interactions of the fusion peptides with membranes can, in large part, explain the differences in the fusogenic activity of the intact protein.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fusión de Membrana/genética , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Deuterio/metabolismo , Ácido Glutámico/genética , Glicina/metabolismo , Cinética , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Isótopos de Fósforo , Conformación Proteica , Estructura Secundaria de Proteína/genética , Serina/genética , Espectroscopía Infrarroja por Transformada de Fourier , Valina/genéticaRESUMEN
Conformational changes occurring in the catalytic cycle of the H+/K+-ATPase were monitored by Fourier transform infrared spectroscopy (FTIR). Caged compounds were used to release ATP, in the presence of Ca2+, to induce the transition between the E1 and E1-P conformation of the H+/K+-ATPase. In addition to bands associated with the photolysis of the caged compounds, some peaks of the difference infrared spectra were associated with changes in secondary structure and modifications of the ionization in the side chains of amino-acid residues (Glu or Asp). These changes were specific to the reaction between the ligand and the enzyme. We estimated that 39 amino acids changed their secondary structure during the reaction and four amino-acid residues were deprotonated. Similar spectral changes appeared when ADP was released from its precursor. The release of Pi from the same caged molecule did not induce similar changes. Changes in tertiary structure occurring during the binding of adenosine and phosphorylation of the enzyme were demonstrated by recording hydrogen/deuterium exchange kinetics by attenuated total reflectance FTIR spectroscopy (ATR-FTIR). At least 129 amide protons were involved in a tertiary structure change induced by ATP. This suggested that secondary structure change transduced a much larger tertiary structure modification.
Asunto(s)
Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Fotólisis , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Espectroscopía Infrarroja por Transformada de Fourier/métodos , PorcinosRESUMEN
A liquid flow cell was used for an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) study of conformational changes taking place in the gastric H+/K+-ATPase. Shifting from E1 to E2 form is induced by replacing Na+ by K+ ions. Introducing ions through a flow passing over a protein multilayer film induced the conformational change without cell manipulations. Measurement sensitivity was thereby improved by about one order of magnitude. The detection threshold allowed the possibility to detect a change affecting five amino acids out of the 1324 that compose the H+/K+-ATPase molecule. It appeared that fewer than five amino-acid residues undergo a conformational change upon replacing Na+ by K+ ions in the medium. Evidence that conformational changes occur in an identical system was brought by monitoring the fluorescence of fluorescein isothiocyanate-labeled H+/K+-ATPase in similar conditions. Our data suggest that essentially the tertiary structure of the protein is modified.
Asunto(s)
ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Dicroismo Circular , Fundus Gástrico/química , Iones , Lípidos/química , Potasio/química , Conformación Proteica , Estructura Secundaria de Proteína , Sodio/química , Espectrometría de Fluorescencia , Espectrofotometría Infrarroja , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , PorcinosRESUMEN
The 120-kDa Na+/Ca2+ exchanger was purified and reconstituted into lipid vesicles. The secondary structure composition of the exchanger was 39% alpha-helices, 20% beta-sheets, 25% beta-turns, and 16% random coils, as analyzed by Fourier transform infrared attenuated total reflection spectroscopy. The secondary structure composition of the COOH-terminal portion of the protein was compatible with a topology model containing 4-6 transmembrane segments. Furthermore, the secondary structure of the NH2-terminal portion of the cytoplasmic loop was analyzed and found to be different from that of the COOH-terminal portion. Ca2+ and/or the exchange inhibitory peptide (XIP) failed to affect the secondary structure of the 120-kDa protein. Tertiary structure modifications induced by Ca2+ and XIP were analyzed by monitoring the hydrogen/deuterium exchange rate for the reconstituted exchanger. In the absence of ligand, 51% of the protein was accessible to solvent. Ca2+ decreased accessibility to 40%, implicating the shielding of at least 103 amino acids. When both Ca2+ and XIP were added, accessibility increased to 66%. No modification was obtained when XIP was added alone. Likewise, in the presence of Ca2+, XIP failed to modify the tertiary structure of the 70-kDa protein, suggesting that XIP acts at the level of the COOH-terminal portion of the intracellular loop. The present data describe, for the first time, conformational changes of the Na+/Ca2+ exchanger induced by Ca2+ and XIP, compatible with an interaction model where regulatory Ca2+ and inhibitory XIP bind to distinct sites, and where XIP binding requires the presence of Ca2+.