 |
PDBsum entry 3ekj
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Fluorescent protein
|
PDB id
|
|
|
|
3ekj
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Crystal structures of the gcamp calcium sensor reveal the mechanism of fluorescence signal change and aid rational design.
|
|
|
Authors
|
|
J.Akerboom,
J.D.Rivera,
M.M.Guilbe,
E.C.Malavé,
H.H.Hernandez,
L.Tian,
S.A.Hires,
J.S.Marvin,
L.L.Looger,
E.R.Schreiter.
|
|
|
Ref.
|
|
J Biol Chem, 2009,
284,
6455-6464.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
Note: In the PDB file this reference is
annotated as "TO BE PUBLISHED". The citation details given above have
been manually determined.
|
|
|
|
Abstract
|
|
|
The genetically encoded calcium indicator GCaMP2 shows promise for neural
network activity imaging, but is currently limited by low signal-to-noise ratio.
We describe x-ray crystal structures as well as solution biophysical and
spectroscopic characterization of GCaMP2 in the calcium-free dark state, and in
two calcium-bound bright states: a monomeric form that dominates at
intracellular concentrations observed during imaging experiments and an
unexpected domain-swapped dimer with decreased fluorescence. This series of
structures provides insight into the mechanism of Ca2+-induced fluorescence
change. Upon calcium binding, the calmodulin (CaM) domain wraps around the M13
peptide, creating a new domain interface between CaM and the circularly permuted
enhanced green fluorescent protein domain. Residues from CaM alter the chemical
environment of the circularly permuted enhanced green fluorescent protein
chromophore and, together with flexible inter-domain linkers, block solvent
access to the chromophore. Guided by the crystal structures, we engineered a
series of GCaMP2 point mutants to probe the mechanism of GCaMP2 function and
characterized one mutant with significantly improved signal-to-noise. The
mutation is located at a domain interface and its effect on sensor function
could not have been predicted in the absence of structural data.
|
|
|
|
|
|
|
|
Figure 3.
Stereoview of the structures of Ca^2^+-saturated GCaMP2-K387W
monomer (red), Ca^2^+-saturated GCaMP2 dimer (yellow), and
Ca^2^+-free 8EF-GCaMP2 (blue) superimposed using the cpEGFP
domain. The proteins are represented as ribbons with the cpEGFP
chromophore represented as sticks.
|
|
Figure 5.
A rationally designed, improved GCaMP2 variant. A,
fluorescence excitation (solid lines) and emission (dashed
lines) spectra of Ca^2+-saturated GCaMP2 T116V (red) and
T116V/D381Y (blue), as well as their calcium-free forms (gray
and black, respectively). Normalized absorbance spectra of each
are shown in the inset. B, close-up stereo view of the
Ca^2+-saturated monomeric GCaMP2 structure, showing the location
of aspartate 381 (D381) of CaM at the CaM/cpEGFP domain
interface. GCaMP2 is displayed as ribbons colored by domain. The
side chain of Asp^381 and the cpEGFP chromophore are displayed
as sticks. A model of a tyrosine side chain at position 381 is
shown in semitransparent sticks to represent a possible
conformation of the D381Y mutant and to illustrate the proximity
of this side chain to the chromophore.
|
|
|
|
|
|
The above figures are
reprinted
from an Open Access publication published by the ASBMB:
J Biol Chem
(2009,
284,
6455-6464)
copyright 2009.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Crystallization and preliminary X-Ray characterization of the genetically encoded fluorescent calcium indicator protein gcamp2
|
|
|
Authors
|
|
M.M rodriguez guilbe,
E.C.Alfaro malave,
J.Akerboom,
J.S.Marvin,
L.L.Looger,
E.R.Schreiter.
|
|
|
Ref.
|
|
Acta Crystallogr ,Sect F, 2008,
64,
629-631.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 3.
Stereoview of the structures of Ca^2^+-saturated GCaMP2-K387W
monomer (red), Ca^2^+-saturated GCaMP2 dimer (yellow), and
Ca^2^+-free 8EF-GCaMP2 (blue) superimposed using the cpEGFP
domain. The proteins are represented as ribbons with the cpEGFP
chromophore represented as sticks.
|
|
Figure 5.
A rationally designed, improved GCaMP2 variant. A,
fluorescence excitation (solid lines) and emission (dashed
lines) spectra of Ca^2+-saturated GCaMP2 T116V (red) and
T116V/D381Y (blue), as well as their calcium-free forms (gray
and black, respectively). Normalized absorbance spectra of each
are shown in the inset. B, close-up stereo view of the
Ca^2+-saturated monomeric GCaMP2 structure, showing the location
of aspartate 381 (D381) of CaM at the CaM/cpEGFP domain
interface. GCaMP2 is displayed as ribbons colored by domain. The
side chain of Asp^381 and the cpEGFP chromophore are displayed
as sticks. A model of a tyrosine side chain at position 381 is
shown in semitransparent sticks to represent a possible
conformation of the D381Y mutant and to illustrate the proximity
of this side chain to the chromophore.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
which is an Open Access publication published by the ASBMB
|
|
|
|
|
|
|
