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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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Normalized fluorescenceresponse curve for tGRP1 (●) and tGRP2 (■). The glucose-responsecurve for tGRP1 and tGRP2 labeled at position 152 (with respect tonative GBP) with MDCC. Data points represent the average of blank-subtractedtriplicate samples. Error bars correspond to ±1 SD.
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fig2: Normalized fluorescenceresponse curve for tGRP1 (●) and tGRP2 (■). The glucose-responsecurve for tGRP1 and tGRP2 labeled at position 152 (with respect tonative GBP) with MDCC. Data points represent the average of blank-subtractedtriplicate samples. Error bars correspond to ±1 SD.

Mentions: The three different proteins were expressed in E. coli and chemically modified via site-selective labelingof a unique Cys residue with a fluorescent coumarin probe, namely,7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin (MDCC).The truncated proteins were characterized in terms of their bindingability to glucose and other sugars. Upon glucose binding, the fluorescenceintensity generated by the MDCC-labeled tGRPs was decreased. Sincethe MDCC-modified Cys is located on a flexible region at the edgeof the binding pocket, we hypothesize that when glucose binds, MDCCis displaced from the binding pocket and oriented more into the solution,thus decreasing its fluorescence. It was found that as the proteinwas increasingly truncated, the apparent KD observed was increased, thus decreasing its affinity for glucose(Figure 2). As more of the stabilizing, second-shellamino acids were removed, the hydrogen-bonding interactions with glucosebecame disrupted, which resulted in an increased KD of 75.2 μM for tGRP1 and 0.25 mM for tGRP2 (Supporting Information, Table 1).


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

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

Normalized fluorescenceresponse curve for tGRP1 (●) and tGRP2 (■). The glucose-responsecurve for tGRP1 and tGRP2 labeled at position 152 (with respect tonative GBP) with MDCC. Data points represent the average of blank-subtractedtriplicate samples. Error bars correspond to ±1 SD.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Normalized fluorescenceresponse curve for tGRP1 (●) and tGRP2 (■). The glucose-responsecurve for tGRP1 and tGRP2 labeled at position 152 (with respect tonative GBP) with MDCC. Data points represent the average of blank-subtractedtriplicate samples. Error bars correspond to ±1 SD.
Mentions: The three different proteins were expressed in E. coli and chemically modified via site-selective labelingof a unique Cys residue with a fluorescent coumarin probe, namely,7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin (MDCC).The truncated proteins were characterized in terms of their bindingability to glucose and other sugars. Upon glucose binding, the fluorescenceintensity generated by the MDCC-labeled tGRPs was decreased. Sincethe MDCC-modified Cys is located on a flexible region at the edgeof the binding pocket, we hypothesize that when glucose binds, MDCCis displaced from the binding pocket and oriented more into the solution,thus decreasing its fluorescence. It was found that as the proteinwas increasingly truncated, the apparent KD observed was increased, thus decreasing its affinity for glucose(Figure 2). As more of the stabilizing, second-shellamino acids were removed, the hydrogen-bonding interactions with glucosebecame disrupted, which resulted in an increased KD of 75.2 μM for tGRP1 and 0.25 mM for tGRP2 (Supporting Information, Table 1).

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