Selective observation of the disordered import signal of a globular protein by in-cell NMR: The example of frataxins
Author(s)
Type
Journal Article
Abstract
We have exploited the capability of in-cell NMR to selectively observe flexible regions withinfolded proteins to carry out a comparative study of two members of the highly conserved frataxin fam-ily which are found both in prokaryotes and in eukaryotes. They all contain a globular domain whichshares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing themitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaYcannot be observed in the homologous E. coli system, although it becomes fully observable as soonas the cells are lysed. This behavior has been observed for several other compact globular proteins asseems to be the rule rather than the exception. The NMR spectrum of the yeast ortholog Yfh1 containsinstead 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 withprevious studies of human frataxin, despite the extensive sequence diversity of this region in the twoproteins. Interestingly, the residues that we observe in in-cell experiments are not visible in the crystalstructure of a Yfh1 mutant design ed to destabilize the first helix. More importantly, our results showthat, in cell, the protein is predominantly present not as an aggregate but as a monomeric species.
Date Issued
2015-04-10
Date Acceptance
2015-03-08
Citation
Protein Science, 2015, 24 (6), pp.996-1003
ISSN
0961-8368
Publisher
Wiley
Start Page
996
End Page
1003
Journal / Book Title
Protein Science
Volume
24
Issue
6
Copyright Statement
© 2015 The Authors Protein Science published by Wiley Periodicals, Inc.on behalf of The Protein Society. This is an open access article under the terms of the CreativeCommons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits use, distributionand reproduction in any medium, provided the original work isproperly cited.
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000355151200008&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemistry & Molecular Biology
flexibility
frataxins
in cell
intrinsically unfolded proteins
NMR
MITOCHONDRIAL PROCESSING PEPTIDASE
MAGNETIC-RESONANCE-SPECTROSCOPY
ESCHERICHIA-COLI
IRON-BINDING
FRIEDREICH ATAXIA
BACTERIAL FRATAXIN
BACKBONE DYNAMICS
YEAST FRATAXIN
CYAY
DIFFUSION
Cytological Techniques
Escherichia coli Proteins
Humans
Intrinsically Disordered Proteins
Iron-Binding Proteins
Magnetic Resonance Spectroscopy
Pliability
0601 Biochemistry And Cell Biology
0802 Computation Theory And Mathematics
0899 Other Information And Computing Sciences
Biophysics
Publication Status
Published