56107 20210121114537.0 doi 10.1016/j.jphotobiol.2015.07.006 sideral 92858 ART-2015-92858 eng Luján, M.A. In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus a-subunit from Synechocystis sp. PCC 6803 2015 Access copy available to the general public Unrestricted 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. info:eu-repo/grantAgreement/ES/DGA/B18 info:eu-repo/grantAgreement/ES/DGA/B33 info:eu-repo/grantAgreement/ES/MICINN/BFU2010-16297 info:eu-repo/grantAgreement/ES/MINECO/AGL2011-23574 info:eu-repo/grantAgreement/ES/MINECO/BIO2012-35271 info:eu-repo/grantAgreement/ES/MINECO/MAT2011-23861 info:eu-repo/semantics/openAccess by-nc-nd http://creativecommons.org/licenses/by-nc-nd/3.0/es/ 3.035 2015 BIOPHYSICS 22 / 72 = 0.306 2015 Q2 T1 BIOCHEMISTRY & MOLECULAR BIOLOGY 119 / 289 = 0.412 2015 Q2 T2 0.723 2015 Biophysics 2015 Q2 Radiology, Nuclear Medicine and Imaging 2015 Q2 Radiological and Ultrasound Technology 2015 Q2 Radiation 2015 Q2 info:eu-repo/semantics/article info:eu-repo/semantics/acceptedVersion (orcid)0000-0002-5406-3280 Martínez, J.I. Universidad de Zaragoza (orcid)0000-0003-3449-4929 Alonso, P.J. Universidad de Zaragoza Torrado, A. Roncel, M. Ortega, J.M. (orcid)0000-0002-2879-9200 Sancho, J. Universidad de Zaragoza Picorel, R. 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 2003 395 Universidad de Zaragoza Dpto. Física Materia Condensa. Área Física Materia Condensada 152 (2015), 308–317 J. photochem. photobiol., B Biol. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 1011-1344 978436 http://zaguan.unizar.es/record/56107/files/texto_completo.pdf Postprint 80603 http://zaguan.unizar.es/record/56107/files/texto_completo.jpg?subformat=icon icon Postprint oai:zaguan.unizar.es:56107 articulos driver 2021-01-21-11:14:33 ARTICLE