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PDBsum entry 2jb5
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Immune system
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PDB id
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2jb5
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References listed in PDB file
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Key reference
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Title
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Fab mor03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-Shielded FAB dimer interface.
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Authors
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R.C.Hillig,
S.Urlinger,
J.Fanghänel,
B.Brocks,
C.Haenel,
Y.Stark,
D.Sülzle,
D.I.Svergun,
S.Baesler,
G.Malawski,
D.Moosmayer,
A.Menrad,
M.Schirner,
K.Licha.
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Ref.
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J Mol Biol, 2008,
377,
206-219.
[DOI no: ]
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PubMed id
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Abstract
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Molecular interactions between near-IR fluorescent probes and specific
antibodies may be exploited to generate novel smart probes for diagnostic
imaging. Using a new phage display technology, we developed such antibody Fab
fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in
vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye
absorption peak of up to 44 nm. This is the largest shift reported for a
biological system so far. Crystal structure determination and absorption
spectroscopy in the crystal in combination with microcalorimetry and small-angle
X-ray scattering in solution revealed that the redshift is triggered by
formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed
protein cavity within the dimer interface. The derived principle of shifting the
absorption peak of a symmetric dye via packaging within a Fab dimer interface
may be transferred to other diagnostic fluorophores, opening the way towards
smart imaging probes that change their wavelength upon interaction with an
antibody.
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Figure 4.
Fig. 4. Crystal structures of the complex of Fab MOR03268:TSC
in two crystal forms. (a) Overall view of the 1:1 complex in
crystal form 1. The Fab molecule is shown in secondary structure
and surface representation, with the heavy chain depicted in
blue and the light chain in orange. TSC (stick representation)
is bound within a deep surface cavity in the antigen-binding
site located at the interface between the two N-terminal
variable immunoglobulin domains (V[L] and V[H]). C[H] and C[L]
refer to the constant immunoglobulin domains of the heavy and
light chain. (b) Stereo representation showing the detailed
binding mode of TSC in form 1. Both the light chain (orange) and
the heavy chain (blue) contribute three loops, the
complementarity determining regions (CDRs). (c) Overall view of
the 2:1 complex in crystal form 2. TSC is bound in the interface
between two Fab molecules, resulting in a TSC-induced Fab dimer
with a very elongated molecular shape. The zoom on the right
side shows a cross section through the Fab dimer interface,
illustrating that the bound TSC molecule site is fully shielded
from the solvent.
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Figure 5.
Fig. 5. SAXS experiments and ab initio SAXS models. (a)
Processed experimental SAXS data (dots with error bars, labeled
from top to bottom): (1) Fab MOR03268, an equimolar Fab–TSC
mixture, a mixture with 1:10 excess of TSC, and the scattering
calculated from the models; (2) monomeric Fab crystal form 1;
(3) compact side-by-side crystallographic Fab dimer observed in
the crystal packing of form 2; (4) elongated Fab dimer from
crystal form 2; (5) a mixture of form 1 and the elongated dimer
from form 2. The plot displays the logarithm of the scattering
intensity I versus momentum transfer s = 4π sin θ/λ, where
2θ is the scattering angle and λ = 1.5 Å is the X-ray
wavelength. The curves are displaced along the abscissa for
clarity. (b) Ab initio SAXS models derived from the data of
TSC-free Fab and of the equimolar Fab–TSC mixture
(semitransparent beads) superimposed with the monomeric crystal
form 1 and the elongated Fab dimer from form 2 (left and right
panels, respectively). Three orthogonal views are displayed.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
377,
206-219)
copyright 2008.
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Secondary reference #1
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Title
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Crystallization and molecular-Replacement solution of a diagnostic fluorescent dye in complex with a specific FAB fragment.
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Authors
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R.C.Hillig,
S.Baesler,
S.Urlinger,
Y.Stark,
S.Bauer,
V.Badock,
M.Huber,
I.Bahr,
M.Schirner,
K.Licha.
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Ref.
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Acta Crystallogr Sect F Struct Biol Cryst Commun, 2007,
63,
217-223.
[DOI no: ]
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PubMed id
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Figure 1.
Structure of TSC and sequence of the Fab molecule MOR03268.
(a) Structure of the fluorescent dye tetrasulfocyanine (TSC).
(b) Amino-acid sequence of the heavy chain of the Fab molecule
MOR03268. The sequence of the C-terminal myc-His[6] tag is shown
in bold. (c) Sequence of the light chain of MOR03268. Acta
Crystallogr Sect F Struct Biol Cryst Commun. 2007 March 1; 63(Pt
3): 217–223. Published online 2007 February 23. doi:
10.1107/S1744309107005957. Copyright [copyright] International
Union of Crystallography 2007
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Figure 4.
Diffraction images. (a) Image of data set 2, crystal form 1,
[Delta][phi] = 1[deg], crystal-to-detector distance 200 mm
(detector edge 2.5 A). (b) Image of data set 4, crystal form 2,
[Delta][phi] = 1[deg], crystal-to-detector distance 220 mm
(detector edge 2.8 A). Despite the very long c axis, the
reflections could still be resolved. Acta Crystallogr Sect F
Struct Biol Cryst Commun. 2007 March 1; 63(Pt 3): 217–223.
Published online 2007 February 23. doi:
10.1107/S1744309107005957. Copyright [copyright] International
Union of Crystallography 2007
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The above figures are
reproduced from the cited reference
which is an Open Access publication published by the IUCr
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