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PDBsum entry 2h3h
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Sugar binding protein
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PDB id
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2h3h
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References listed in PDB file
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Key reference
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Title
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Structure-Based design of robust glucose biosensors using a thermotoga maritima periplasmic glucose-Binding protein.
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Authors
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Y.Tian,
M.J.Cuneo,
A.Changela,
B.Höcker,
L.S.Beese,
H.W.Hellinga.
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Ref.
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Protein Sci, 2007,
16,
2240-2250.
[DOI no: ]
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PubMed id
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Abstract
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We report the design and engineering of a robust, reagentless fluorescent
glucose biosensor based on the periplasmic glucose-binding protein obtained from
Thermotoga maritima (tmGBP). The gene for this protein was cloned from genomic
DNA and overexpressed in Escherichia coli, the identity of its cognate sugar was
confirmed, ligand binding was studied, and the structure of its glucose complex
was solved to 1.7 Angstrom resolution by X-ray crystallography. TmGBP is
specific for glucose and exhibits high thermostability (midpoint of thermal
denaturation is 119 +/- 1 degrees C and 144 +/- 2 degrees C in the absence and
presence of 1 mM glucose, respectively). A series of fluorescent conjugates was
constructed by coupling single, environmentally sensitive fluorophores to unique
cysteines introduced by site-specific mutagenesis at positions predicted to be
responsive to ligand-induced conformational changes based on the structure.
These conjugates were screened to identify engineered tmGBPs that function as
reagentless fluorescent glucose biosensors. The Y13C*Cy5 conjugate is bright,
gives a large response to glucose over concentration ranges appropriate for in
vivo monitoring of blood glucose levels (1-30 mM), and can be immobilized in an
orientation-specific manner in microtiter plates to give a reversible response
to glucose. The immobilized protein retains its response after long-term storage
at room temperature.
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Figure 2.
Figure 2. Crystal structure of tmGBP. (A) The overall
structure complexed with glucose (ball and stick) including
sites of cysteine mutations for fluorophore attachment (magenta
spheres). (B) Close-up view of the binding pocket: (green:
glucose; dashed line: hydrogen bonds). The figure was generated
using MOLSCRIPT (Kraulis 1991) and RASTER3D (Merritt and
Murphy1994).
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Figure 6.
Figure 6. Successive cycles of glucose titration and buffer
washes of tmGBP-Y13C-Czif immobilized on microtiter plates (20
mM MOPS and 150 mM NaCl, pH 7.0, 25°C). Data of apo and
saturated ligand binding states for five cycles are connected by
straight lines.
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The above figures are
reprinted
by permission from the Protein Society:
Protein Sci
(2007,
16,
2240-2250)
copyright 2007.
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