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A new trend to determine biochemical parameters by quantitative FRET assays.

Liao JY, Song Y, Liu Y - Acta Pharmacol. Sin. (2015)

Bottom Line: Historically, FRET assays have been used to quantitatively deduce molecular distances.In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m).We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.

ABSTRACT
Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

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Related in: MedlinePlus

Design of FRET-based protease assay. Schematic depicting the CyPet-(pre-SUMO)-YPet substrate indicating the principle of FRET from CyPet (donor) to YPet (acceptor).
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fig3: Design of FRET-based protease assay. Schematic depicting the CyPet-(pre-SUMO)-YPet substrate indicating the principle of FRET from CyPet (donor) to YPet (acceptor).

Mentions: The general strategy for a FRET-based protease assay is based on a fluorescent protein-tagged substrate (Figure 3). The SENP substrate, pre-SUMOs, is flanked by a FRET pair, CyPet and YPet. The fused recombinant protein is cleaved by the protease SENPs to release two products: the CyPet-SUMO and the SUMO tail with YPet, which leads to the disruption of FRET and an increase in the emission of CyPet while dramatical decrease in the emission of YPet after the excitation of CyPet. The decreased fluorescent emission of YPet after cleavage can be used to characterize the kinetic properties of SENPs in real time.


A new trend to determine biochemical parameters by quantitative FRET assays.

Liao JY, Song Y, Liu Y - Acta Pharmacol. Sin. (2015)

Design of FRET-based protease assay. Schematic depicting the CyPet-(pre-SUMO)-YPet substrate indicating the principle of FRET from CyPet (donor) to YPet (acceptor).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4816234&req=5

fig3: Design of FRET-based protease assay. Schematic depicting the CyPet-(pre-SUMO)-YPet substrate indicating the principle of FRET from CyPet (donor) to YPet (acceptor).
Mentions: The general strategy for a FRET-based protease assay is based on a fluorescent protein-tagged substrate (Figure 3). The SENP substrate, pre-SUMOs, is flanked by a FRET pair, CyPet and YPet. The fused recombinant protein is cleaved by the protease SENPs to release two products: the CyPet-SUMO and the SUMO tail with YPet, which leads to the disruption of FRET and an increase in the emission of CyPet while dramatical decrease in the emission of YPet after the excitation of CyPet. The decreased fluorescent emission of YPet after cleavage can be used to characterize the kinetic properties of SENPs in real time.

Bottom Line: Historically, FRET assays have been used to quantitatively deduce molecular distances.In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m).We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.

ABSTRACT
Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.

Show MeSH
Related in: MedlinePlus