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Glucose recognition proteins for glucose sensing at physiological concentrations and temperatures.

Joel S, Turner KB, Daunert S - ACS Chem. Biol. (2014)

Bottom Line: The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins.The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability.This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami , 1011 NW 15th Street, Miami, Florida 33136, United States.

ABSTRACT
Advancements in biotechnology have allowed for the preparation of designer proteins with a wide spectrum of unprecedented chemical and physical properties. A variety of chemical and genetic methods can be employed to tailor the protein's properties, including its stability and various functions. Herein, we demonstrate the production of semisynthetic glucose recognition proteins (GRPs) prepared by truncating galactose/glucose binding protein (GBP) of E. coli and expanding the genetic code via global incorporation of unnatural amino acids into the structure of GBP and its fragments. The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins. The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability. These modifications enabled the utilization of the protein for the detection of glucose within physiological concentrations (mM) and temperatures ranging from hypothermia to hyperthermia. This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

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Far-UV CD absorbance of (A. GBP H152C (black),tGRP1 (red), and tGRP2 (blue). (B) uGRPFW (green), uGRP1FW (red),and uGRP2FW (blue). (C) uGRPFL (green), uGRP1FL (red), and uGRP2FL(blue). Three accumulations were averaged for each sample at RT. Theresponse for each blank was subtracted from the response for the correspondingsample, and the resulting spectra are shown.
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fig5: Far-UV CD absorbance of (A. GBP H152C (black),tGRP1 (red), and tGRP2 (blue). (B) uGRPFW (green), uGRP1FW (red),and uGRP2FW (blue). (C) uGRPFL (green), uGRP1FL (red), and uGRP2FL(blue). Three accumulations were averaged for each sample at RT. Theresponse for each blank was subtracted from the response for the correspondingsample, and the resulting spectra are shown.

Mentions: Further characterization of thetGRPs by circular dichroism revealed that, similar to native GBP,there was little change in the overall secondary structure characteristicsof the proteins. This is evident from the far-UV CD absorbance spectrafor GBP152, tGRP1, and tGRP2 shown in Figure 5a. The α-helix peak (approximately 210 and 220 nm) for GBPH152C is much intense than that of tGRPs showing a loss of α-helicalstructure on truncation.


Glucose recognition proteins for glucose sensing at physiological concentrations and temperatures.

Joel S, Turner KB, Daunert S - ACS Chem. Biol. (2014)

Far-UV CD absorbance of (A. GBP H152C (black),tGRP1 (red), and tGRP2 (blue). (B) uGRPFW (green), uGRP1FW (red),and uGRP2FW (blue). (C) uGRPFL (green), uGRP1FL (red), and uGRP2FL(blue). Three accumulations were averaged for each sample at RT. Theresponse for each blank was subtracted from the response for the correspondingsample, and the resulting spectra are shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215909&req=5

fig5: Far-UV CD absorbance of (A. GBP H152C (black),tGRP1 (red), and tGRP2 (blue). (B) uGRPFW (green), uGRP1FW (red),and uGRP2FW (blue). (C) uGRPFL (green), uGRP1FL (red), and uGRP2FL(blue). Three accumulations were averaged for each sample at RT. Theresponse for each blank was subtracted from the response for the correspondingsample, and the resulting spectra are shown.
Mentions: Further characterization of thetGRPs by circular dichroism revealed that, similar to native GBP,there was little change in the overall secondary structure characteristicsof the proteins. This is evident from the far-UV CD absorbance spectrafor GBP152, tGRP1, and tGRP2 shown in Figure 5a. The α-helix peak (approximately 210 and 220 nm) for GBPH152C is much intense than that of tGRPs showing a loss of α-helicalstructure on truncation.

Bottom Line: The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins.The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability.This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami , 1011 NW 15th Street, Miami, Florida 33136, United States.

ABSTRACT
Advancements in biotechnology have allowed for the preparation of designer proteins with a wide spectrum of unprecedented chemical and physical properties. A variety of chemical and genetic methods can be employed to tailor the protein's properties, including its stability and various functions. Herein, we demonstrate the production of semisynthetic glucose recognition proteins (GRPs) prepared by truncating galactose/glucose binding protein (GBP) of E. coli and expanding the genetic code via global incorporation of unnatural amino acids into the structure of GBP and its fragments. The unnatural amino acids 5,5,5-trifluoroleucine (FL) and 5-fluorotryptophan (FW) were chosen for incorporation into the proteins. The resulting semisynthetic GRPs exhibit enhanced thermal stability and increased detection range of glucose without compromising its binding ability. These modifications enabled the utilization of the protein for the detection of glucose within physiological concentrations (mM) and temperatures ranging from hypothermia to hyperthermia. This ability to endow proteins such as GBP with improved stability and properties is critical in designing the next generation of tailor-made biosensing proteins for continuous in vivo glucose monitoring.

Show MeSH
Related in: MedlinePlus