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

¹ Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA
² Department of Pharmacology and Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA

(RECEIVED April 25, 2007; FINAL REVISION April 25, 2007; ACCEPTED June 12, 2007)

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 Å 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.

Keywords: glucose-binding protein; structure; thermostable; fluorescent biosensor

Abbreviations: PBP, periplasmic binding protein; tmGBP, Thermotoga maritima glucose-binding protein; ecRBP, Escherichia coli ribose-binding protein; ecGBP, E. coli glucose-binding protein; ttGBP, Thermus thermophilus glucose-binding protein; ecMBP, E. coli maltose-binding protein; ORF, open reading frame; CD, circular dichroism; GuHCl, guanidine hydrogen chloride; IANBD, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine; Zif, zinc finger domain; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; IMAC, immobilized metal affinity chromatography; SV, Stern-Volmer.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072969407.

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