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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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Structuresof the protein and the unnatural amino acid analogues incorporatedinto the proteins. (A) Native GBP with glucose in the ligand-bindingpocket and Ca2+ present in the Ca2+-bindingpocket (PDB ID: 2GBP). (B) GBP’s binding pocket is magnified, showing Cys152 andthe amino acids involved in H-bonding with glucose. (C) tGRP1- (D),tGRP2-, and (E) tGRP3-truncated areas of the original GBP proteinare shown in red. (F) 5,5,5-Trifluoroleucine. (G) 5-Fluorotryptophan.
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fig1: Structuresof the protein and the unnatural amino acid analogues incorporatedinto the proteins. (A) Native GBP with glucose in the ligand-bindingpocket and Ca2+ present in the Ca2+-bindingpocket (PDB ID: 2GBP). (B) GBP’s binding pocket is magnified, showing Cys152 andthe amino acids involved in H-bonding with glucose. (C) tGRP1- (D),tGRP2-, and (E) tGRP3-truncated areas of the original GBP proteinare shown in red. (F) 5,5,5-Trifluoroleucine. (G) 5-Fluorotryptophan.

Mentions: Reliable, low cost technologiesfor glucose sensing has been the focus of a continuous active fieldof research since the first glucose biosensing device was proposedin 1962.1,2 Despite recent technological improvementsin consumer devices, current-generation commercially available glucosemeters still measure glucose by employing electrochemical detectionbased on traditional enzymes, such as glucose oxidase (GOx) or glucose-1-dehydrogenase(GDH).1 These electrochemical methods exhibitpoor performance in the hypoglycemic range and suffer from hematocritdependence and interference from electrochemically active molecules,hypoxemia, or hypotension.1 Lack of selectivityis especially troublesome, as the list of interfering compounds includesmolecules commonly found in blood such as acetaminophen, salicylicacid, ibuprofen, ascorbic acid, etc.2 Toovercome these limitations, alternative technologies based on an opticalresponse using rationally engineered glucose sensing proteins arebeing explored. To that end, research is being aimed at achievingreagentless optical sensing systems that are sensitive, selective,reproducible, accurate, rugged, and capable of glucose detection atphysiological concentrations and temperatures. Among new methods developedare those based on fluorescence,3−5 fluorescence resonance energytransfer (FRET),3,5 and bioluminescence.6 These optical methods exploit the hinge-motionconformational change exhibited by the glucose/galactose binding protein(GBP) (Figure 1), a periplasmic binding proteinfound in many bacteria that undergoes a conformational change uponbinding glucose. GBP has been extensively studied as a possible sensingcomponent of future generations of devices for the continuous, real-timemonitoring of lucose for the management of diabetes.3,4,6,7


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

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

Structuresof the protein and the unnatural amino acid analogues incorporatedinto the proteins. (A) Native GBP with glucose in the ligand-bindingpocket and Ca2+ present in the Ca2+-bindingpocket (PDB ID: 2GBP). (B) GBP’s binding pocket is magnified, showing Cys152 andthe amino acids involved in H-bonding with glucose. (C) tGRP1- (D),tGRP2-, and (E) tGRP3-truncated areas of the original GBP proteinare shown in red. (F) 5,5,5-Trifluoroleucine. (G) 5-Fluorotryptophan.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215909&req=5

fig1: Structuresof the protein and the unnatural amino acid analogues incorporatedinto the proteins. (A) Native GBP with glucose in the ligand-bindingpocket and Ca2+ present in the Ca2+-bindingpocket (PDB ID: 2GBP). (B) GBP’s binding pocket is magnified, showing Cys152 andthe amino acids involved in H-bonding with glucose. (C) tGRP1- (D),tGRP2-, and (E) tGRP3-truncated areas of the original GBP proteinare shown in red. (F) 5,5,5-Trifluoroleucine. (G) 5-Fluorotryptophan.
Mentions: Reliable, low cost technologiesfor glucose sensing has been the focus of a continuous active fieldof research since the first glucose biosensing device was proposedin 1962.1,2 Despite recent technological improvementsin consumer devices, current-generation commercially available glucosemeters still measure glucose by employing electrochemical detectionbased on traditional enzymes, such as glucose oxidase (GOx) or glucose-1-dehydrogenase(GDH).1 These electrochemical methods exhibitpoor performance in the hypoglycemic range and suffer from hematocritdependence and interference from electrochemically active molecules,hypoxemia, or hypotension.1 Lack of selectivityis especially troublesome, as the list of interfering compounds includesmolecules commonly found in blood such as acetaminophen, salicylicacid, ibuprofen, ascorbic acid, etc.2 Toovercome these limitations, alternative technologies based on an opticalresponse using rationally engineered glucose sensing proteins arebeing explored. To that end, research is being aimed at achievingreagentless optical sensing systems that are sensitive, selective,reproducible, accurate, rugged, and capable of glucose detection atphysiological concentrations and temperatures. Among new methods developedare those based on fluorescence,3−5 fluorescence resonance energytransfer (FRET),3,5 and bioluminescence.6 These optical methods exploit the hinge-motionconformational change exhibited by the glucose/galactose binding protein(GBP) (Figure 1), a periplasmic binding proteinfound in many bacteria that undergoes a conformational change uponbinding glucose. GBP has been extensively studied as a possible sensingcomponent of future generations of devices for the continuous, real-timemonitoring of lucose for the management of diabetes.3,4,6,7

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