Sensors 2013, 13(4), 4553-4570; https://doi.org/10.3390/s130404553
Internal Calibration Förster Resonance Energy Transfer Assay: A Real-Time Approach for Determining Protease Kinetics
1
School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang, China
2
Department of Bioengineering, Center for Bioengineering Research, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA
3
Institute for Integrative Genome Biology, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA
4
Keck Proteomics Laboratory, Institute for Integrative Genome Biology, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA
‡
Current address: California Institute of Biomedical Research, 11119 N Torrey Pines Rd, La Jolla, CA, 92037, USA.
§
Current address: Department of Bioengineering, University of Pennsylvania, 3451 Walnut Street, Philadelphia, PA 19104, USA.
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Received: 18 February 2013 / Revised: 11 March 2013 / Accepted: 25 March 2013 / Published: 8 April 2013
(This article belongs to the Special Issue Enzymatic Biosensors)
Abstract
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research. This powerful tool can elucidate protein interactions in either a dynamic or steady state. We recently developed a series of FRET-based technologies to determine protein interaction dissociation constant and for use in high-throughput screening assays of SUMOylation. SUMO (small ubiquitin-like modifier) is conjugated to substrates through an enzymatic cascade. This important posttranslational protein modification is critical for multiple biological processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate SUMO from its substrate. Here, we describe a novel quantitative FRET-based protease assay for determining the kinetics of SENP1. Our strategy is based on the quantitative analysis and differentiation of fluorescent emission signals at the FRET acceptor emission wavelengths. Those fluorescent emission signals consist of three components: the FRET signal and the fluorescent emissions of donor (CyPet) and acceptor (YPet). Unlike our previous method in which donor and acceptor direct emissions were excluded by standard curves, the three fluorescent emissions were determined quantitatively during the SENP digestion process from onesample. New mathematical algorithms were developed to determine digested substrate concentrations directly from the FRET signal and donor/acceptor direct emissions. The kinetic parameters, kcat, KM, and catalytic efficiency (kcat/KM) of SENP1 catalytic domain for pre-SUMO1/2/3 were derived. Importantly, the general principles of this new quantitative methodology of FRET-based protease kinetic determinations can be applied to other proteases in a robust and systems biology approach. View Full-TextKeywords:
quantitative FRET analysis; internal calibration; one-sample assay; protease kinetics; SENP
This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).
Share & Cite This Article
MDPI and ACS Style
Jiang, L.; Liu, Y.; Song, Y.; Saavedra, A.N.; Pan, S.; Xiang, W.; Liao, J. Internal Calibration Förster Resonance Energy Transfer Assay: A Real-Time Approach for Determining Protease Kinetics. Sensors 2013, 13, 4553-4570.