Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor.
Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.
Amiss
Terry J
TJ
BD Technologies, Research Triangle Park, NC 27709, USA. Terry_Amiss@bd.com
Sherman
Douglas B
DB
Nycz
Colleen M
CM
Andaluz
Sandra A
SA
Pitner
J Bruce
JB
D001665:Binding Sites; D015374:Biosensing Techniques; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D015723:Gene Library; D005947:Glucose / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008968:Molecular Conformation; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*
D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D005947:Glucose