English   español  
Por favor, use este identificador para citar o enlazar a este item: http://hdl.handle.net/10261/121093
Compartir / Impacto:
Título : In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803
Autor : Luján Serrano, María Ángeles ; Martínez Jesús I.; Alonso, Pablo J.; Torrado, Alejandro; Roncel Gil, Mercedes ; Ortega, José M. ; Sancho, Javier ; Picorel Castaño, Rafael
Palabras clave : Alpha-helix
Cytochrome
Membrane
Photosynthesis
Chimeric protein
Protein assembly
Fecha de publicación : 2015
Citación : In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus α-subunit from Synechocystis sp. PCC 6803. Journal of Photochemistry and Photobiology B: Biology 152: 308-317 (2015).
Resumen: The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, α and β. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the β-subunit (β/β) but not with the full length α-subunit. In the present work, reconstitution of a homodimer (α/α) cytochrome b559 like structure was possible using a chimeric N-terminus α-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic α-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the α-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric α-subunit cytochrome b559 like structure. The features of the new structure were determined by UV–Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane.
Descripción : 25 Pags.- 9 Figs.- The definitive version is available at: http://www.sciencedirect.com/science/journal/10111344
Versión del editor: http://dx.doi.org/10.1016/j.jphotobiol.2015.07.006
URI : http://hdl.handle.net/10261/121093
DOI: 10.1016/j.jphotobiol.2015.07.006
ISSN: 1011-1344
Aparece en las colecciones: (EEAD) Artículos
(IQFR) Artículos
Ficheros en este ítem:
Fichero Descripción Tamaño Formato  
PicorelR_JPhotochemPhotobiolB_2015.pdf1,02 MBAdobe PDFVista previa
Visualizar/Abrir
Mostrar el registro completo
 



NOTA: Los ítems de Digital.CSIC están protegidos por copyright, con todos los derechos reservados, a menos que se indique lo contrario.