TY  - JOUR
A1  - Ikeya, Teppei
A1  - Hanashima, Tomomi
A1  - Hosoya, Saori
A1  - Shimazaki, Manato
A1  - Ikeda, Shiro
A1  - Mishima, Masaki
A1  - Güntert, Peter
A1  - Ito, Yutaka
T1  - Improved in-cell structure determination of proteins at near-physiological concentration
T2  - Scientific reports
N2  - Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells.
KW  - Biopolymers in vivo
KW  - Computational models
KW  - Data processing
KW  - Solution-state NMR
Y1  - 2016
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/46576
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-465762
SN  - 2045-2322
N1  - This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
VL  - 6
IS  - Art. 38312
SP  - 1
EP  - 11
PB  - Macmillan Publishers Limited, part of Springer Nature
CY  - [London]
ER  -