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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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Thermal denaturation curves for (A) GBP H152C,(B) tGRP1 (red) and tGRP2 (blue), (C) uGRPFL (red) and uGRPFW (blue),(D) uGRP1FW (red) and uGRP2FW (blue), (E) uGRP1FL (red) and uGRP2FL(blue). (F) Table showing the melting tempratures of proteins. Proteinswere prepared at a concentration of 0.2 mg mL–1 inbuffer (10 mM phosphate, 0.2 mM CaCl2, pH = 7.5). Alphahelix denaturation was monitored by CD at 222 nm as the temperaturewas increased from 10 to 70 °C. All Tm values are the average of ±0.1 to ±0.6 SD (n = 2).
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fig4: Thermal denaturation curves for (A) GBP H152C,(B) tGRP1 (red) and tGRP2 (blue), (C) uGRPFL (red) and uGRPFW (blue),(D) uGRP1FW (red) and uGRP2FW (blue), (E) uGRP1FL (red) and uGRP2FL(blue). (F) Table showing the melting tempratures of proteins. Proteinswere prepared at a concentration of 0.2 mg mL–1 inbuffer (10 mM phosphate, 0.2 mM CaCl2, pH = 7.5). Alphahelix denaturation was monitored by CD at 222 nm as the temperaturewas increased from 10 to 70 °C. All Tm values are the average of ±0.1 to ±0.6 SD (n = 2).

Mentions: To determine whetherthe overall structural stability of the tGRPs had been indeed affected,the thermal stability of each tGRP and GBP H152C was determined bycircular dichroism (CD) spectroscopy. Protein thermal stability isan important consideration when developing protein-based sensors thatwill be used for extended periods of time at 37 °C, the temperatureof the human body. Improved thermal stability should increase thelifetime of the sensor, allowing for long-term, reproducible glucosedetermination. GBP H152C has a melting temperature (Tm) of 52.4 °C Figure 4a. Truncatingthe native structure had a significant effect on the thermal stability.A decrease in the Tm was observed as thesize of the protein decreased after truncation (Figure 4). Since tGRP3 showed no glucose response, it was not furthercharacterized. We hypothesized that this drastic loss in thermal stabilityreflects the instability of the protein structure as a result of truncation,which likely contributes to the increase in dissociation constantsfor its ligand, glucose.


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

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

Thermal denaturation curves for (A) GBP H152C,(B) tGRP1 (red) and tGRP2 (blue), (C) uGRPFL (red) and uGRPFW (blue),(D) uGRP1FW (red) and uGRP2FW (blue), (E) uGRP1FL (red) and uGRP2FL(blue). (F) Table showing the melting tempratures of proteins. Proteinswere prepared at a concentration of 0.2 mg mL–1 inbuffer (10 mM phosphate, 0.2 mM CaCl2, pH = 7.5). Alphahelix denaturation was monitored by CD at 222 nm as the temperaturewas increased from 10 to 70 °C. All Tm values are the average of ±0.1 to ±0.6 SD (n = 2).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Thermal denaturation curves for (A) GBP H152C,(B) tGRP1 (red) and tGRP2 (blue), (C) uGRPFL (red) and uGRPFW (blue),(D) uGRP1FW (red) and uGRP2FW (blue), (E) uGRP1FL (red) and uGRP2FL(blue). (F) Table showing the melting tempratures of proteins. Proteinswere prepared at a concentration of 0.2 mg mL–1 inbuffer (10 mM phosphate, 0.2 mM CaCl2, pH = 7.5). Alphahelix denaturation was monitored by CD at 222 nm as the temperaturewas increased from 10 to 70 °C. All Tm values are the average of ±0.1 to ±0.6 SD (n = 2).
Mentions: To determine whetherthe overall structural stability of the tGRPs had been indeed affected,the thermal stability of each tGRP and GBP H152C was determined bycircular dichroism (CD) spectroscopy. Protein thermal stability isan important consideration when developing protein-based sensors thatwill be used for extended periods of time at 37 °C, the temperatureof the human body. Improved thermal stability should increase thelifetime of the sensor, allowing for long-term, reproducible glucosedetermination. GBP H152C has a melting temperature (Tm) of 52.4 °C Figure 4a. Truncatingthe native structure had a significant effect on the thermal stability.A decrease in the Tm was observed as thesize of the protein decreased after truncation (Figure 4). Since tGRP3 showed no glucose response, it was not furthercharacterized. We hypothesized that this drastic loss in thermal stabilityreflects the instability of the protein structure as a result of truncation,which likely contributes to the increase in dissociation constantsfor its ligand, glucose.

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