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Engineering and rapid selection of a low‐affinity glucose/galactose‐binding protein for a glucose biosensor
Author(s) -
Amiss Terry J.,
Sherman Douglas B.,
Nycz Colleen M.,
Andaluz Sandra A.,
Pitner J. Bruce
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073119507
Subject(s) - periplasmic space , biosensor , chemistry , biochemistry , escherichia coli , dissociation constant , fluorophore , galactose , glucose transporter , protein engineering , saturated mutagenesis , mutant , biophysics , fluorescence , biology , enzyme , insulin , receptor , quantum mechanics , gene , endocrinology , physics
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 μM 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.