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Frontiers in Biology

Front Biol    2012, Vol. 7 Issue (1) : 57-64
Quantitative analysis of FRET assay in biology New developments in protein interaction affinity and protease kinetics determinations in the SUMOylation cascade
Yan LIU, Yang SONG, Ling JIANG, Jiayu LIAO(email.png)
Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA
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F?rster resonance energy transfer (FRET) techniques have been widely used in biological studies in vitro and in vivo and are powerful tools for elucidating protein interactions in many regulatory cascades. FRET occurs between oscillating dipoles of two fluorophores with overlapping emission and excitation wavelengths and is dependent on the spectroscopic and geometric properties of the donor-acceptor pair. Various efforts have been made to develop quantitative FRET methods to accurately determine the interaction affinity and kinetics parameters. SUMOylation is an important post-translational protein modification with key roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENP) act as endopeptidases to process the pre-SUMO or an isopeptidase to deconjugate SUMO from its substrate. Here we also summarize recent developments of theoretical and experimental procedures for determining the protein interaction dissociation constant, Kd, and protease kinetics parameters, kcat and Km, in the SUMOylation pathway. The general principles of these quantitative FRET-based measurements can be applied to other protein interactions and proteases.

Keywords quantitative FRET analysis      protein affinity determination      kinetics analysis     
Corresponding Author(s): LIAO Jiayu,   
Issue Date: 01 February 2012
 Cite this article:   
Yan LIU,Jiayu LIAO,Yang SONG, et al. Quantitative analysis of FRET assay in biology New developments in protein interaction affinity and protease kinetics determinations in the SUMOylation cascade[J]. Front Biol, 2012, 7(1): 57-64.
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Jiayu LIAO
Fig.1  SUMOylation conjugation cascade. ① Maturation of SUMO by cleavage of SUMO C-terminus by SENP proteases. ② SUMO activation (linkage to E1) by heterodimer of E1 ligase. ③ SUMO is transferred to E2 ligase. ④ Conjugation of SUMO peptide to substrate(s) is mediated by E3 ligase. ⑤ Removal of SUMO peptide from substrate by SENP proteases. Please see the text for abbreviations.
Fig.2  Quantitative analysis of fluorescence signals of FRET emission. RFU: Relative fluorescent units. (A) Fluorescent emission at acceptor wavelength (530 nm) () can be divided into three fractions—FRET emission, direct emission of donor, and direct emission of acceptor. (B) Acceptor emission when excited at 475 nm (). (C) Donor emission at 475 nm () and 530 nm () when excited at 414 nm. (D) Donor emission at 530 nm when excited at 414 nm () or 475 nm ().
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