Structure-Based Design of Robust Glucose Biosensors using a Thermotoga maritima Periplasmic Glucose-Binding Protein
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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 959969
- Report Number(s):
- BNL-82955-2009-JA; TRN: US201016%%1113
- Journal Information:
- Protein Science, Vol. 16
- Country of Publication:
- United States
- Language:
- English
Similar Records
Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima
PEGylation of a Maltose Biosensor Promotes Enhanced Signal Response
Related Subjects
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