PDBsum entry 2h3h

Sugar binding protein

PDB id: 2h3h

Contents

Protein chains

313 a.a. *

Ligands

BGC ×2

Waters ×668

* Residue conservation analysis

PDB id:

2h3h

Name:

Sugar binding protein

Title:

Crystal structure of the liganded form of thermotoga maritima glucose binding protein

Structure:

Sugar abc transporter, periplasmic sugar-binding protein. Chain: a, b. Engineered: yes

Source:

Thermotoga maritima. Organism_taxid: 2336. Gene: tm0114. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.70Å R-factor: 0.205 R-free: 0.241

Authors:

A.Changela,Y.Tian

Key ref:

Y.Tian et al. (2007). Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein. Protein Sci, 16, 2240-2250. PubMed id: 17766373 DOI: 10.1110/ps.072969407

Date:

22-May-06 Release date: 22-May-07

Protein chains

Q9WXW9  (Q9WXW9_THEMA) -  Sugar ABC transporter, periplasmic sugar-binding protein from Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8)

Seq: 335 a.a.

Struc: 313 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1110/ps.072969407

Protein Sci 16:2240-2250 (2007)

PubMed id: 17766373

Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein.

Y.Tian, M.J.Cuneo, A.Changela, B.Höcker, L.S.Beese, H.W.Hellinga.

ABSTRACT

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.

Selected figure(s)

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).

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.

The above figures are reprinted by permission from the Protein Society: Protein Sci (2007, 16, 2240-2250) copyright 2007.

Figures were selected by an automated process.