Improved in-cell structure determination of proteins at near-physiological concentration

  • 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.

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Author:Teppei Ikeya, Tomomi Hanashima, Saori Hosoya, Manato Shimazaki, Shiro Ikeda, Masaki Mishima, Peter Güntert, Yutaka Ito
URN:urn:nbn:de:hebis:30:3-465762
DOI:https://doi.org/10.1038/srep38312
ISSN:2045-2322
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/27910948
Parent Title (English):Scientific reports
Publisher:Macmillan Publishers Limited, part of Springer Nature
Place of publication:[London]
Document Type:Article
Language:English
Year of Completion:2016
Date of first Publication:2016/12/02
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2018/06/05
Tag:Biopolymers in vivo; Computational models; Data processing; Solution-state NMR
Volume:6
Issue:Art. 38312
Page Number:11
First Page:1
Last Page:11
Note:
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/
HeBIS-PPN:433822708
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Wissenschaftliche Zentren und koordinierte Programme / Zentrum für Biomolekulare Magnetische Resonanz (BMRZ)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Sammlung Biologie / Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung 4.0