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PDBsum entry 3evr
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Signaling protein
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PDB id
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3evr
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References listed in PDB file
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Key reference
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Title
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Structural basis for calcium sensing by gcamp2.
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Authors
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Q.Wang,
B.Shui,
M.I.Kotlikoff,
H.Sondermann.
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Ref.
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Structure, 2008,
16,
1817-1827.
[DOI no: ]
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PubMed id
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Note: In the PDB file this reference is
annotated as "TO BE PUBLISHED". The citation details given above have
been manually determined.
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Abstract
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Genetically encoded Ca(2+) indicators are important tools that enable the
measurement of Ca(2+) dynamics in a physiologically relevant context. GCaMP2,
one of the most robust indicators, is a circularly permutated EGFP
(cpEGFP)/M13/calmodulin (CaM) fusion protein that has been successfully used for
studying Ca(2+) fluxes in vivo in the heart and vasculature of transgenic mice.
Here we describe crystal structures of bright and dim states of GCaMP2 that
reveal a sophisticated molecular mechanism for Ca(2+) sensing. In the bright
state, CaM stabilizes the fluorophore in an ionized state similar to that
observed in EGFP. Mutational analysis confirmed critical interactions between
the fluorophore and elements of the fused peptides. Solution scattering studies
indicate that the Ca(2+)-free form of GCaMP2 is a compact, predocked state,
suggesting a molecular basis for the relatively rapid signaling kinetics
reported for this indicator. These studies provide a structural basis for the
rational design of improved Ca(2+)-sensitive probes.
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Figure 1.
Figure 1. Crystal Structures of GCaMP2•Ca^2+ and cpEGFP
(A) Domain organization of GCaMP2 and truncated
derivatives. A schematic presentation of the GCaMP2 fusion
protein is shown. The color scheme introduced here is maintained
throughout the article. Residue numbering for circularly
permutated EGFP (cpEGFP) and GCaMP2ΔRSET follows the sequence
of GCaMP2.
(B) Crystal structure of the isolated cpEGFP
moiety. The C-terminal fragment of C-EGFP is colored in light
green, and the N-terminal fragment is colored in dark green. Two
orthogonal views are shown.
(C) Crystal structure of
monomeric GCaMP2ΔRSET in its Ca^2+-bound state. Crystals were
grown in the presence of 1 mM Ca^2+. Two orthogonal views are
shown. The M13 helix is shown in blue, and the calmodulin (CaM)
domain is shown in red. The cpEFGP is colored as described in
(B).
(D) Comparison of crystal structures of GCaMP2,
cpEGFP, and GFP-S65T. Distance difference matrices based on Cα
positions were used to compare the conformation of cpEGFP in
isolation (bottom-right triangle) and as part of GCaMP2
(top-left triangle) with the structure of GFP-S65T (PDB code
1EMA; see Supplemental Experimental Procedures). Difference
matrices were regularized using a Z-score analysis and
color-coded accordingly. Each entry in the matrix depicts the
difference in distance between corresponding Cα atoms in the
two structures. Distances that show little change are blue. Red
entries represent distances that are significantly different in
the two structures.
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Figure 3.
Figure 3. Intramolecular Interfaces in Monomeric
GCaMP2•Ca^2+
(A) Interfaces among the cpEGFP, M13, and
CaM modules in the structure of monomeric GCaMP2ΔRSET•Ca^2+.
Residues of the M13-cpEGFP module interacting with CaM are
colored red. Interfacial residues on CaM are colored in green
and blue for contacts with cpEGFP and the M13 helix,
respectively. A top view, rotated 90° around the horizontal
axis with respect to the view shown above, is shown as a cutaway
rendition of the surface (bottom-left). The fluorophores of
cpEGFP and Arg-377 of CaM are shown in stick presentation.
Surface presentation of the isolated CaM domain and M13-cpEGFP
unit were rotated by +90° and −90°, respectively, with
respect to the view of the assembled structure (top-left).
(B) Electrostatic potential of the M13-cpEGFP module and CaM
mapped onto its molecular surface. Views are identical to (A).
Red represents negative and blue represents positive potential
(−5 to +5 k[B]T).
(C) Schematic diagram of the
fluorophore environment and the hydrogen bond network between
cpEGFP and CaM. The numbering scheme for GCaMP2 was used.
Corresponding residue numbers in GFP are shown in brackets.
Carbon atoms of residues in cpEGFP, CaM, and linker segments are
shown in green, dark red, and gray, respectively. Hydrogen bonds
shown in the figure are between 2.7 and 3.3 Å (not drawn
to scale).
(D) Close-up views of the interfacial regions in
GCaMP2ΔRSET•Ca^2+. Water-mediated interaction between the
fluorophore and Arg-377 of the CaM domain (top) and cpEGFP:CaM
interfacial residues (bottom) are shown.
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The above figures are
reprinted
from an Open Access publication published by Cell Press:
Structure
(2008,
16,
1817-1827)
copyright 2008.
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