RESUMEN
Ligand-dependent changes in accessibility of purified P-glycoprotein, functionally reconstituted in liposomes, were investigated by fluorescence measurements. Trp quenching experiments provided evidence that P-glycoprotein adopts different tertiary structures upon binding of drug substrates in the absence and presence of MgATP and its nonhydrolyzable analog, MgATPgammaS. Five anthracycline derivatives were tested as drug substrates: daunorubicin, 4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and methoxy-morpholino-doxorubicin. Among them, daunorubicin and 4'-epi-doxorubicin have been shown to be rejected outside the multidrug-resistant cells, whereas the three others have been shown to accumulate in multidrug-resistant cells overexpressing P-glycoprotein and therefore retain their cytotoxic activity. A small conformational change was associated with nucleotide binding and amplified after nucleotide hydrolysis. Different conformational states were adopted by P-glycoprotein upon the addition of the anthracycline derivatives in the absence and presence of MgATP or MgATPgammaS. These conformational changes are shown to be related to the nature of the antitumor agents and more precisely to their capacity to accumulate in resistant cells. These data also suggest that the cytotoxicity of iododoxorubicin and 4-demethoxy-daunorubicin is related to the fact they are not transported by P-glycoprotein. On the contrary, methoxy-morpholino-doxorubicin cytotoxicity may be explained in terms of its rapid reincorporation into the plasma membrane after being transported by P-glycoprotein.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Ligandos , Estructura Terciaria de Proteína , Acrilamida/farmacología , Adenosina Trifosfato/farmacología , Marcadores de Afinidad , Animales , Antibióticos Antineoplásicos/farmacología , Azidas , Células CHO , Cricetinae , Dihidropiridinas , Resistencia a Múltiples Medicamentos , Fluorescencia , Nucleótidos/metabolismo , Unión Proteica , Conformación Proteica , Proteolípidos/química , Triptófano/químicaRESUMEN
Hydrophobins are small fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes that, in the case of Class I hydrophobins, can be disassembled only by treatment with agents like pure trifluoroacetic acid. Here we characterize, by spectroscopic techniques, the structural changes that occur upon assembly at an air/water interface and upon assembly on a hydrophobic solid surface, and the influence of deglycosylation on these events. We determined that the hydrophobin SC3 from Schizophyllum commune contains 16-22 O-linked mannose residues, probably attached to the N-terminal part of the peptide chain. Scanning force microscopy revealed that SC3 adsorbs specifically to a hydrophobic surface and cannot be removed by heating at 100 degrees C in 2% sodium dodecyl sulfate. Attenuated total reflection Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed that the monomeric, water-soluble form of the protein is rich in beta-sheet structure and that the amount of beta-sheet is increased after self-assembly on a water-air interface. Alpha-helix is induced specifically upon assembly of the protein on a hydrophobic solid. We propose a model for the formation of rodlets, which may be induced by dehydration and a conformational change of the glycosylated part of the protein, resulting in the formation of an amphipathic alpha-helix that forms an anchor for binding to a substrate. The assembly in the beta-sheet form seems to be involved in lowering of the surface tension, a potential function of hydrophobins.
Asunto(s)
Proteínas Fúngicas/química , Adsorción , Aire , Fenómenos Biofísicos , Biofisica , Dicroismo Circular , Glicosilación , Manosa/química , Microscopía de Fuerza Atómica , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Schizophyllum/química , Espectroscopía Infrarroja por Transformada de Fourier , Tensión Superficial , AguaRESUMEN
The structure of purified P-glycoprotein functionally reconstituted into liposomes was investigated by attenuated total reflection Fourier transform infrared spectroscopy. A quantitative evaluation of the secondary structure and a kinetic of 2H/H exchange of the P-glycoprotein were performed both in the presence and in the absence of MgATP, MgATP-verapamil, and MgADP. This approach was previously shown to be a useful tool to detect tertiary structure changes resulting from the interaction between a protein and its specific ligands, as established for the Neurospora crassa H+-ATPase. 2H/H exchange measurements provided evidence that a large fraction of the P-glycoprotein is poorly accessible to the aqueous medium. Addition of MgATP induced an increased accessibility to the solvent of a population of amino acids, while addition of MgATP-verapamil resulted in a subtraction of a part of the protein from access to the aqueous solvent. No significant changes were observed upon addition of MgADP or verapamil alone. The secondary structure of P-glycoprotein was not affected by addition of ligands. The variations observed in the 2H/H exchange rate when P-glycoprotein interacted with the above ligands therefore represented tertiary structure changes. Fluorescence quenching experiments confirmed that MgATP-induced changes are to be found in the tertiary structure of the enzyme.
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/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico , Daunorrubicina/metabolismo , Deuterio , Cinética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteolípidos/metabolismo , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Hepatitis B surface antigen particles are composed of the major viral envelope protein, the S protein, embedded into a lipid shell. The description of the folding of this protein within the particle membrane could provide helpful information for replacing surface-exposed protein domains by foreign sequences without destabilizing the particle structure. Since the crystallization of the protein in its lipid environment remains inaccessible in the near future, alternative approaches had to be envisaged. We combine here the available experimental structural and topological data with a conformational procedure to identify membrane-associated domains of the HBsAg protein and to propose a three-dimensional description of their assembly within the particle membrane. The proposed protein structure is composed of four membrane-spanning helices and an amphipatic helix located on the inner surface membrane. The transmembrane helices are assembled into a highly hydrophobic complex in which no access to the water environment is allowed. The approach could be extended to other membrane-associated proteins.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/química , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Datos de Secuencia Molecular , Conformación Proteica , Proteínas del Envoltorio Viral/químicaRESUMEN
HBsAg particles are highly immunogenic and have been shown to be a suitable support for the presentation of foreign epitopes. More information about the topology of the HBsAg protein is a prerequisite to any rational attempt to replace the region of this protein with foreign epitopes without modifying the assembly of the particles. This topology and, more precisely, the mode of interaction of the HBsAg protein with the lipid will depend on the lipid organization in the particle envelope. Nothing is known concerning the lipid organization of HBsAg particles. The only available information concerns their lipid composition. Phospholipase D hydrolysis of HBsAg particles was used here to determine whether the particles were surrounded with a lipid monolayer or bilayer. The lipid fluidity within the particle envelope was evaluated by fluorescence polarization measurements. The data strongly suggest that the HBsAg particle membrane is organized as a discontinuous rigid bilayer of lipids interacting with protein aggregates.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/química , Virus de la Hepatitis B/química , Membrana Dobles de Lípidos/química , Proteínas Virales/química , Difenilhexatrieno/análogos & derivados , Difenilhexatrieno/química , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Antígenos de Superficie de la Hepatitis B/ultraestructura , Humanos , Hidrólisis , Fosfatidilcolinas , Fosfolipasa D/metabolismo , Conformación Proteica , Proteínas Virales/ultraestructuraRESUMEN
Hepatitis B surface antigen particles are highly immunogenic and have been shown to provide a suitable support for the presentation of foreign epitopes. More information about the topology of their constitutive protein, the S (small envelope) protein, is a prerequisite to any rational attempt to replace region of this protein with foreign epitopes without modifying the assembly of the particle. The topology of the S protein within the lipid membrane was investigated here by combining extensive proteolysis of the external protein domains with proteinase K and (FTIR-ATR). The proteolytic hydrolysis of the S protein and the identification of the digestion products allowed characterization of the membrane-protected regions of the protein. FTIR spectra of the digested hepatitis B particles revealed that the peptides associated with the particles are rich in alpha-helix structure. The kinetic of 2H/H exchange provided evidence that a large fraction of the native S protein is poorly accessible to the aqueous medium.