title: Selective observation of the disordered import signal of a globular protein by in-cell NMR: the example of frataxins.
abstract: We have exploited the capability of in-cell NMR to selectively observe flexible regions within folded proteins to carry out a comparative study of two members of the highly conserved frataxin family which are found both in prokaryotes and in eukaryotes. They all contain a globular domain which shares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing the mitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaY cannot be observed in the homologous E. coli system, although it becomes fully observable as soon as the cells are lysed. This behavior has been observed for several other compact globular proteins as seems to be the rule rather than the exception. The NMR spectrum of the yeast ortholog Yfh1 contains instead visible signals from the protein. We demonstrate that they correspond to the flexible N-terminal tail indicating that this is flexible and unfolded. This flexibility of the N-terminus agrees with previous studies of human frataxin, despite the extensive sequence diversity of this region in the two proteins. Interestingly, the residues that we observe in in-cell experiments are not visible in the crystal structure of a Yfh1 mutant designed to destabilize the first helix. More importantly, our results show that, in cell, the protein is predominantly present not as an aggregate but as a monomeric species.
authors.affiliation: National Institute for Medical Research, MRC, The Ridgeway, London, United Kingdom.
authors.forename: Matija
authors.initials: M
authors.lastname: Popovic
authors.affiliation: Department of Clinical Neuroscience, King's College London, Denmark Hill Campus, London, United Kingdom.
authors.forename: Domenico
authors.initials: D
authors.lastname: Sanfelice
authors.affiliation: Department of Life Sciences, Centre for Structural Biology, Sir Ernst Chain Building, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Chiara
authors.initials: C
authors.lastname: Pastore
authors.affiliation: Department of Life Sciences, Centre for Structural Biology, Sir Ernst Chain Building, Imperial College London, London, SW7 2AZ, United Kingdom.
authors.forename: Filippo
authors.initials: F
authors.lastname: Prischi
authors.affiliation: National Institute for Medical Research, MRC, The Ridgeway, London, United Kingdom.
authors.forename: Piero Andrea
authors.initials: PA
authors.lastname: Temussi
authors.affiliation: Department of Clinical Neuroscience, King's College London, Denmark Hill Campus, London, United Kingdom.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Pastore
mesh_terms: D003584:Cytological Techniques; D029968:Escherichia coli Proteins; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry* / Q000378:metabolism*; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D018583:Pliability; D000096923:Frataxin
chemical_list: D029968:Escherichia coli Proteins; D064267:Intrinsically Disordered Proteins; D033862:Iron-Binding Proteins; D000096923:Frataxin; C120906:CyaY protein, E coli
keywords: NMR; flexibility; frataxins; in cell; intrinsically unfolded proteins