Protein structure determination in living cells by in-cell NMR spectroscopy
Yutaka Ito1,2,3, Daisuke Sakakibara1,2, Atsuko Sasaki1,2, Teppei Ikeya1,4, Junpei Hamatsu1,2,Tomomi Hanashima1, Masaki Mishima1,2, Tsutomu Mikawa3, Markus Wälchli5, Brian O.
Smith6, Masahiro Shirakawa2,7 and Peter Güntert1,41

Department of Chemistry, Tokyo Metropolitan University, Japan; 2CREST/JST; 3ResearchGroup for Bio-supramolecular Structure-Function, RIKEN; 4Institute of BiophysicalChemistry, J. W. Goethe-University Frankfurt, Germany; 5Bruker BioSpin, Japan; 6Divisionof Biochemistry and Molecular Biology, University of Glasgow, UK; 7Department ofMolecular Engineering, Kyoto University, Japan.
Introduction
In vivo observations of three-dimensional structures, structural changes, dynamics orinteractions of proteins are essential for the explicit understanding of the structural basis of
their functions inside cells. The non-invasive character and its ability to provide data atatomic resolution make NMR spectroscopy ideally suited for the task. Despite the interest inobserving hetronuclear multi-dimensional NMR of macromolecules in living cells (in-cellNMR), it has not yet been confirmed whether the existing NMR methods for the structure
determination of purified proteins can be applicable to proteins in living cells.
Results and Discussion
We use as a model system theThermus thermophilus HB8
TTHA1718 gene product, aputative heavy metal binding
protein consisting of 66 aminoacids that was overexpressed inE. coli cells to a concentration of3−4 mM. The stability of thelive E. coli samples were
examined under measurement
conditions and confirmed thatTTHA1718 in-cell NMR samples are stable for at least 6 hours(Figure 1a).
First we performed backbone
resonance assignment of TTHA1718 in E. coli cells using six 3D
triple-resonance NMR spectra. Figure 1. Experimental procedures for in-cell NMR
We applied a nonlinear sampling experiments using E. coli cells. a, Scheme for checking thescheme for the indirectly ac- stability of E. coli cells under NMR measurement conditions.
b, Rapid acquisition of 3D NMR spectra by employing aquired dimensions, which has nonlinear sampling scheme. The 2D maximum entropy
been shown in combination with method was used for processing nonlinearly sampled
maximum entropy processing to dimensions.
provide significant time savingsin the measurement of multidimensional NMR experiments (Figure 1b). All backbone resonances of the non-N-terminal
and non-proline residues in TTHA1718 were assigned except for three residues in thehypothetical metal binding loop predicted from primary sequence analysis. Similarly weassigned the majority of sidechain resonances by analysing
three additional 3D tripleresonance NMR spectra.
Next, we performed NMR
experiments of TTHA1718 in E.
coli cells with selectively 1H/13Clabelled methyl groups of Ala,Leu and Val, aiming at obtaining
unambiguous long-range distance restraints involving methylgroups. Further NOE-derived
distance restraints were obtained
from 3D 15N-separated NOESY
and 3D 13C-separated NOESY
spectra measured on uniformly Figure 2. NMR solution structure of TTHA1718 in livinglabelled E.coli samples.
E. coli cells. a, A superposition of the 20 final structures ofThe structure calculation of TTHA1718 in living E. coli cells, showing the backbone (N, Cα,TTHA1718 in E. coli cells was C’) atoms. b, A superposition of the 20 final structures ofperformed with the program purified TTHA1718 in vitro. c, A superposition of the 20 final
CYANA v3.0 on the basis of structures of TTHA1718 in living E. coli cells calculated
without distance restraints derived from NOEs involving methyl
NOE-derived distance restraints, groups obtained in methyl-selectively protonated in-cell NMR
TALOS-derived backbone tor- samples. d, Secondary structure of TTHA1718 in living E.
sion angle restraints, and re- coli cells. The sidechains of Ala, Leu and Val residues whosestraints for hydrogen bonds in methyl groups were labelled with 1H/13C are shown. e.
α-helices and β-sheets. The re- Distance restraints derived from methyl group-correlated andsulting structure is well con- other NOEs are represented in the ribbon model with thick andthin lines, respectively.
verged with a backbone RMSDof 0.96 Å to the mean coordinates (Figure 2a), and is very similar to the structure that was determined independently invitro from a purified TTHA1718 sample (Figure 2b). The backbone RMSD between thein-cell and in vitro structures is 1.16 Å.
This result strongly suggests that high resolution 3D structures of proteins can be
determined in solution in the cytoplasm of bacterial cells. Rapid data measurement using
nonlinear sampling scheme with maximum entropy reconstruction, which provided spectra ofmuch higher quality than conventional spectra recorded in the same short measurement time,and selective protonation at methyl groups (Figure 2d and 2e) to enable the observation of
long-range unambiguous NOEs were key for the success of this study (Figure 2c).
By extending this approach it will become possible to study the conformations of proteinsin detail and how they change in response to biological events in living environments. In
particular, this approach provides the tools that will permit the effects of molecular crowdingin the cytosol and the conformations of intrinsically disordered proteins to be investigated inliving cells.