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Protein interaction affinity determination by quantitative FRET technology
Author(s) -
Song Yang,
Rodgers V.G.J.,
Schultz Jerome S.,
Liao Jiayu
Publication year - 2012
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.24564
Subject(s) - förster resonance energy transfer , chemistry , dissociation constant , sumo protein , protein–protein interaction , isothermal titration calorimetry , covalent bond , biophysics , combinatorial chemistry , fluorescence , biochemistry , ubiquitin , organic chemistry , physics , receptor , quantum mechanics , biology , gene
The dissociation constant, K d , is an important parameter for characterizing protein–protein interaction affinities. SUMOylation is one of the important protein post‐translational modifications and it involves a multi‐step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non‐covalent protein–protein interactions are involved in this cascade. Techniques involving Förster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the K d by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant ( K d ) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 4–40, the K d s of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET‐based K d determination approach. This technology, therefore, can be used in general for protein–protein interaction dissociation constant determination. Biotechnol. Bioeng. 2012; 109: 2875–2883. © 2012 Wiley Periodicals, Inc.