In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus a-subunit from Synechocystis sp. PCC 6803
Resumen: The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, a and ß. 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 ß-subunit (ß/. ß) but not with the full length a-subunit. In the present work, reconstitution of a homodimer (a/. a) cytochrome b559 like structure was possible using a chimeric N-terminus a-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic a-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 a-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 a-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.
Idioma: Inglés
DOI: 10.1016/j.jphotobiol.2015.07.006
Año: 2015
Publicado en: JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 152 (2015), 308–317
ISSN: 1011-1344

Factor impacto JCR: 3.035 (2015)
Categ. JCR: BIOPHYSICS rank: 22 / 72 = 0.306 (2015) - Q2 - T1
Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 119 / 289 = 0.412 (2015) - Q2 - T2

Factor impacto SCIMAGO:

Financiación: info:eu-repo/grantAgreement/ES/DGA/B18
Financiación: info:eu-repo/grantAgreement/ES/DGA/B33
Financiación: info:eu-repo/grantAgreement/ES/MICINN/BFU2010-16297
Financiación: info:eu-repo/grantAgreement/ES/MINECO/AGL2011-23574
Financiación: info:eu-repo/grantAgreement/ES/MINECO/BIO2012-35271
Financiación: info:eu-repo/grantAgreement/ES/MINECO/MAT2011-23861
Tipo y forma: Article (PostPrint)
Área (Departamento): Bioquímica y Biología Molecular (Departamento de Bioquímica y Biología Molecular y Celular)

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Exportado de SIDERAL (2017-05-03-14:31:32)

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 Record created 2016-07-12, last modified 2017-05-03


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