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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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Normalizedfluorescence response curve for uGRPs with (A) FW and (B) FL. Glucose-responsecurves with globally incorporated FW and FL labeled at position 152(with respect to native GBP) with MDCC for uGRP (▲), uGRP1(●), and uGRP2 (■). Data points represent the averageof blank-subtracted triplicate samples. Error bars correspond to ±1SD.
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fig6: Normalizedfluorescence response curve for uGRPs with (A) FW and (B) FL. Glucose-responsecurves with globally incorporated FW and FL labeled at position 152(with respect to native GBP) with MDCC for uGRP (▲), uGRP1(●), and uGRP2 (■). Data points represent the averageof blank-subtracted triplicate samples. Error bars correspond to ±1SD.

Mentions: Theglucose binding ability of the uGRPs was evaluated. Dose–responsecurves for glucose generated with the uGRPs with FW and FL, respectively,are shown in Figure 6. The percent fluorescencequenching of uGRPs labeled with MDCCin solution containing 10–3 M glucose is shown in SupportingInformation, Table 3. Glucose binding was maintained with theGRPs containing unnatural amino acids. Apparent KD’s were determined to be 2.02 × 10–6 M, 4.1 × 10–6 M, 7.5 × 10–4 M, 5.4 × 10–5 M, 1.9 × 10–4 M and 1.8 × 10–4 M for uGRP-FW, uGRP1-FW,uGRP2-FW, uGRP-FL, uGRP1-FL and uGRP2-FL, respectively (Supporting Information, Table 1). It was observedthat proteins with unnatural tryptophan demonstrated lower detectionlimits compared to the proteins with unnatural leucines. In orderto further characterize the structural changes resulting from unnaturalamino acid incorporation and the corresponding effects on binding,far-UV CD analysis was carried out. The far-UV CD spectrum of uGRP-FWwas similar to the CD spectrum of GBP152, indicating that the secondarystructure of GBP was not significantly altered by incorporation ofunnatural tryptophans (Figure 5b). This wasfurther supported by the apparent KD’sof both GBP H152C4 and uGRP-FW in the micromolarrange. However, the CD spectrum of uGRP-FL (Figure 5c) was different from that of GBP152, as the peaks at 222and 208 nm were less intense for uGRP-FL. This suggested that thesecondary structure of GBP152 was altered by incorporating unnaturalleucines. This may explain the change in apparent KD as compared to GBP H152C. The CD spectrum of uGRP1/2-FW(Figure 5b) revealed that the incorporationof fluorinated tryptophans in the trunctated proteins, i.e., tGRP1/2,altered the secondary structure of the protein as compared to thefull length GBP-FW (uGRP-FW). This was explained again by both theposition and intensity of peaks at 222 and 208 nm. The peak at 222nm for tGRP1/2-FW was less pronounced than the corresponding peakfor uGRP-FW, while the peak at 208 nm in uGRP-FW was shifted towardlower wavelength (204 nm) in tGRP1/2-FW. Also, when comparing thesecondary structure of tGRP1/2-FW with that of tGRP1/2, it was evidentthat the peak around 208 nm, as seen in both the tGRP1/2, is shiftedto a lower wavelength of 204 nm in tGRP1/2-FW, suggesting a decreasein alpha helical content. However, the CD spectrum of tGRP1/2-FL (Figure 5c) suggests that the incorporation of fluorinatedleucines in tGRP1/2 alters the secondary structure when compared totGRP1/2, as evident from the difference in the intensities of peaks.


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

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

Normalizedfluorescence response curve for uGRPs with (A) FW and (B) FL. Glucose-responsecurves with globally incorporated FW and FL labeled at position 152(with respect to native GBP) with MDCC for uGRP (▲), uGRP1(●), and uGRP2 (■). Data points represent the averageof blank-subtracted triplicate samples. Error bars correspond to ±1SD.
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fig6: Normalizedfluorescence response curve for uGRPs with (A) FW and (B) FL. Glucose-responsecurves with globally incorporated FW and FL labeled at position 152(with respect to native GBP) with MDCC for uGRP (▲), uGRP1(●), and uGRP2 (■). Data points represent the averageof blank-subtracted triplicate samples. Error bars correspond to ±1SD.
Mentions: Theglucose binding ability of the uGRPs was evaluated. Dose–responsecurves for glucose generated with the uGRPs with FW and FL, respectively,are shown in Figure 6. The percent fluorescencequenching of uGRPs labeled with MDCCin solution containing 10–3 M glucose is shown in SupportingInformation, Table 3. Glucose binding was maintained with theGRPs containing unnatural amino acids. Apparent KD’s were determined to be 2.02 × 10–6 M, 4.1 × 10–6 M, 7.5 × 10–4 M, 5.4 × 10–5 M, 1.9 × 10–4 M and 1.8 × 10–4 M for uGRP-FW, uGRP1-FW,uGRP2-FW, uGRP-FL, uGRP1-FL and uGRP2-FL, respectively (Supporting Information, Table 1). It was observedthat proteins with unnatural tryptophan demonstrated lower detectionlimits compared to the proteins with unnatural leucines. In orderto further characterize the structural changes resulting from unnaturalamino acid incorporation and the corresponding effects on binding,far-UV CD analysis was carried out. The far-UV CD spectrum of uGRP-FWwas similar to the CD spectrum of GBP152, indicating that the secondarystructure of GBP was not significantly altered by incorporation ofunnatural tryptophans (Figure 5b). This wasfurther supported by the apparent KD’sof both GBP H152C4 and uGRP-FW in the micromolarrange. However, the CD spectrum of uGRP-FL (Figure 5c) was different from that of GBP152, as the peaks at 222and 208 nm were less intense for uGRP-FL. This suggested that thesecondary structure of GBP152 was altered by incorporating unnaturalleucines. This may explain the change in apparent KD as compared to GBP H152C. The CD spectrum of uGRP1/2-FW(Figure 5b) revealed that the incorporationof fluorinated tryptophans in the trunctated proteins, i.e., tGRP1/2,altered the secondary structure of the protein as compared to thefull length GBP-FW (uGRP-FW). This was explained again by both theposition and intensity of peaks at 222 and 208 nm. The peak at 222nm for tGRP1/2-FW was less pronounced than the corresponding peakfor uGRP-FW, while the peak at 208 nm in uGRP-FW was shifted towardlower wavelength (204 nm) in tGRP1/2-FW. Also, when comparing thesecondary structure of tGRP1/2-FW with that of tGRP1/2, it was evidentthat the peak around 208 nm, as seen in both the tGRP1/2, is shiftedto a lower wavelength of 204 nm in tGRP1/2-FW, suggesting a decreasein alpha helical content. However, the CD spectrum of tGRP1/2-FL (Figure 5c) suggests that the incorporation of fluorinatedleucines in tGRP1/2 alters the secondary structure when compared totGRP1/2, as evident from the difference in the intensities of peaks.

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