2h3h
From Proteopedia
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| - | + | {{STRUCTURE_2h3h| PDB=2h3h | SCENE= }} | |
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'''Crystal structure of the liganded form of Thermotoga maritima glucose binding protein''' | '''Crystal structure of the liganded form of Thermotoga maritima glucose binding protein''' | ||
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| + | ==Overview== | ||
| + | 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. | ||
==About this Structure== | ==About this Structure== | ||
2H3H is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Thermotoga_maritima Thermotoga maritima]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2H3H OCA]. | 2H3H is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Thermotoga_maritima Thermotoga maritima]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2H3H OCA]. | ||
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| + | ==Reference== | ||
| + | Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein., Tian Y, Cuneo MJ, Changela A, Hocker B, Beese LS, Hellinga HW, Protein Sci. 2007 Oct;16(10):2240-50. Epub 2007 Aug 31. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/17766373 17766373] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Thermotoga maritima]] | [[Category: Thermotoga maritima]] | ||
[[Category: Changela, A.]] | [[Category: Changela, A.]] | ||
[[Category: Tian, Y.]] | [[Category: Tian, Y.]] | ||
| - | [[Category: | + | [[Category: Gbp]] |
| - | [[Category: | + | [[Category: Glucose binding protein]] |
| - | [[Category: | + | [[Category: Periplasmic binding protein]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Apr 16 23:06:26 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 20:06, 16 April 2008
Crystal structure of the liganded form of Thermotoga maritima glucose binding protein
Overview
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.
About this Structure
2H3H is a Single protein structure of sequence from Thermotoga maritima. Full crystallographic information is available from OCA.
Reference
Structure-based design of robust glucose biosensors using a Thermotoga maritima periplasmic glucose-binding protein., Tian Y, Cuneo MJ, Changela A, Hocker B, Beese LS, Hellinga HW, Protein Sci. 2007 Oct;16(10):2240-50. Epub 2007 Aug 31. PMID:17766373 Page seeded by OCA on Wed Apr 16 23:06:26 2008
