 |
PDBsum entry 2roe
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Metal binding protein
|
PDB id
|
|
|
|
2roe
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Protein structure determination in living cells by in-Cell nmr spectroscopy.
|
|
|
Authors
|
|
D.Sakakibara,
A.Sasaki,
T.Ikeya,
J.Hamatsu,
T.Hanashima,
M.Mishima,
M.Yoshimasu,
N.Hayashi,
T.Mikawa,
M.Wälchli,
B.O.Smith,
M.Shirakawa,
P.Güntert,
Y.Ito.
|
|
|
Ref.
|
|
Nature, 2009,
458,
102-105.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Investigating proteins 'at work' in a living environment at atomic resolution is
a major goal of molecular biology, which has not been achieved even though
methods for the three-dimensional (3D) structure determination of purified
proteins in single crystals or in solution are widely used. Recent developments
in NMR hardware and methodology have enabled the measurement of high-resolution
heteronuclear multi-dimensional NMR spectra of macromolecules in living cells
(in-cell NMR). Various intracellular events such as conformational changes,
dynamics and binding events have been investigated by this method. However, the
low sensitivity and the short lifetime of the samples have so far prevented the
acquisition of sufficient structural information to determine protein structures
by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure
calculated exclusively on the basis of information obtained in living cells. The
structure of the putative heavy-metal binding protein TTHA1718 from Thermus
thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell
NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the
indirectly acquired dimensions was used to overcome problems caused by the
instability and low sensitivity of living E. coli samples. Almost all of the
expected backbone NMR resonances and most of the side-chain NMR resonances were
observed and assigned, enabling high quality (0.96 ångström backbone root mean
squared deviation) structures to be calculated that are very similar to the in
vitro structure of TTHA1718 determined independently. The in-cell NMR approach
can thus provide accurate high-resolution structures of proteins in living
environments.
|
|
|
|
|
|
|
|
Figure 2.
Figure 2: Rapid acquisition of 3D NMR spectra of TTHA1718 in
living E. coli cells. a, Rapid acquisition of 3D NMR spectra
using a nonlinear sampling scheme. DFT, discrete Fourier
transform; dim, dimension; MaxEnt, maximum-entropy processing.
b, Repeated observation of 3D NMR spectra with intermittent
monitoring of the sample condition by short 2D ^1H–^15N HSQC
experiments.
|
|
Figure 3.
Figure 3: Collection of NOE-derived distance restraints in
TTHA1718 in living E. coli cells. a, Methyl region of the
^1H–^13C heteronuclear multiple-quantum coherence (HMQC)
spectrum of the selectively methyl-protonated sample.
Assignments of the methyl groups of Ala, Leu and Val residues
are indicated, if available. b, ^13C–^13C cross-sections
corresponding to the ^1H frequencies of representative methyl
groups extracted from the 3D ^13C/^13C-separated HMQC-NOE-HMQC
spectrum. The cross-peaks due to interresidual NOEs are assigned
in red. Intraresidual NOEs are indicated by blue boxes and
annotated. c, ^1H–^1H cross-sections corresponding to the ^15N
frequencies of selected backbone amide groups extracted from the
3D ^15N-separated NOESY-HSQC spectrum. The inter- and
intraresidue NOEs are indicated as in b. d, Topology diagram of
the  -sheet
structure in TTHA1718. Interstrand backbone NOEs are depicted as
double-headed arrows.
|
|
|
|
|
|
The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nature
(2009,
458,
102-105)
copyright 2009.
|
|
|
|
|
|
|
