The authors declare that they have no competing financial interests.
Protein interaction affinity determination by quantitative FRET technology†
Article first published online: 18 JUN 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 109, Issue 11, pages 2875–2883, November 2012
How to Cite
Song, Y., Rodgers, V.G.J., Schultz, J. S. and Liao, J. (2012), Protein interaction affinity determination by quantitative FRET technology. Biotechnol. Bioeng., 109: 2875–2883. doi: 10.1002/bit.24564
- Issue published online: 24 SEP 2012
- Article first published online: 18 JUN 2012
- Manuscript Accepted: 15 MAY 2012
- Manuscript Revised: 27 APR 2012
- Manuscript Received: 22 MAR 2012
- National Institutes of Health. Grant Number: 5 R01 AI076504
- fluorescent proteins;
- quantitative FRET;
- Kd determination;
The dissociation constant, Kd, 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 Kd 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 (Kd) 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 Kds 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 Kd 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.