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Title: Structure-Based Design of Robust Glucose Biosensors using a Thermotoga maritima Periplasmic Glucose-Binding Protein

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 Angstroms resolution by X-ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 {+-} 1 C and 144 {+-} 2 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 aftermore » long-term storage at room temperature.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959969
Report Number(s):
BNL-82955-2009-JA
TRN: US201016%%1113
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Protein Science; Journal Volume: 16
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; BLOOD; CONFORMATIONAL CHANGES; CRYSTALLOGRAPHY; DESIGN; DNA; ESCHERICHIA COLI; GENES; GLUCOSE; IN VIVO; MONITORING; MUTAGENESIS; PLATES; PROTEINS; RESOLUTION; SACCHAROSE; STORAGE; national synchrotron light source

Citation Formats

Tian,Y., Cunco, M., Changela, A., Hocker, B., Beese, L., and Hellinga, H.. Structure-Based Design of Robust Glucose Biosensors using a Thermotoga maritima Periplasmic Glucose-Binding Protein. United States: N. p., 2007. Web. doi:10.1110/ps.072969407.
Tian,Y., Cunco, M., Changela, A., Hocker, B., Beese, L., & Hellinga, H.. Structure-Based Design of Robust Glucose Biosensors using a Thermotoga maritima Periplasmic Glucose-Binding Protein. United States. doi:10.1110/ps.072969407.
Tian,Y., Cunco, M., Changela, A., Hocker, B., Beese, L., and Hellinga, H.. Mon . "Structure-Based Design of Robust Glucose Biosensors using a Thermotoga maritima Periplasmic Glucose-Binding Protein". United States. doi:10.1110/ps.072969407.
@article{osti_959969,
title = {Structure-Based Design of Robust Glucose Biosensors using a Thermotoga maritima Periplasmic Glucose-Binding Protein},
author = {Tian,Y. and Cunco, M. and Changela, A. and Hocker, B. and Beese, L. and Hellinga, H.},
abstractNote = {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 Angstroms resolution by X-ray crystallography. TmGBP is specific for glucose and exhibits high thermostability (midpoint of thermal denaturation is 119 {+-} 1 C and 144 {+-} 2 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.},
doi = {10.1110/ps.072969407},
journal = {Protein Science},
number = ,
volume = 16,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}