title: Functional expression of the PorAH channel from Corynebacterium glutamicum in cell-free expression systems: implications for the role of the naturally occurring mycolic acid modification.
abstract: PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. (15)N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties.
authors.affiliation: Centre National de la Recherche Scientifique, CNRS UMR 5089, Institut de Pharmacologie et de Biologie Structurale, 205 Route de Narbonne, Toulouse F-31077, Cedex 04, France.
authors.forename: Parthasarathi
authors.initials: P
authors.lastname: Rath
authors.forename: Pascal
authors.initials: P
authors.lastname: Demange
authors.forename: Olivier
authors.initials: O
authors.lastname: Saurel
authors.forename: Marielle
authors.initials: M
authors.lastname: Tropis
authors.forename: Mamadou
authors.initials: M
authors.lastname: Daffé
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ghazi
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D048230:Corynebacterium glutamicum; D004926:Escherichia coli; D015870:Gene Expression; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D009171:Mycolic Acids / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011499:Protein Processing, Post-Translational
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D009171:Mycolic Acids