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Selective observation of the disordered import signal of a globular protein by in-cell NMR: The example of frataxins

Popovic, Matija and Sanfelice, Domenico and Pastore, Chiara and Prischi, Filippo and Temussi, Piero Andrea and Pastore, Annalisa (2015) 'Selective observation of the disordered import signal of a globular protein by in-cell NMR: The example of frataxins.' Protein Science, 24 (6). pp. 996-1003. ISSN 0961-8368

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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.

Item Type: Article
Uncontrolled Keywords: flexibility; frataxins; in cell; intrinsically unfolded proteins; NMR
Subjects: Q Science > Q Science (General)
Divisions: Faculty of Science and Health
Faculty of Science and Health > Life Sciences, School of
SWORD Depositor: Elements
Depositing User: Elements
Date Deposited: 18 Dec 2015 13:14
Last Modified: 15 Jan 2022 00:30

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