Abstract
Knowledge of the kinetics of protein—protein interactions has become important in defining nuclear receptor function. Such knowledge allows characterization of interactions that occur with high affinity and/or selectivity. Surface plasmon resonance is a useful and sensitive tool for studying protein—protein interactions. This technique involves immobilization of a “ligand” to the surface of a sensor chip and subsequently passing over multiple concentrations of “analyte” to generate binding curves. Interaction between the receptor and target protein is monitored by the density at the sensor chip surface and allows calculation of the association and dissociation stages (and therefore affinity) of interactions to be assessed in real-time. Using software packages, these kinetic parameters can be quantified. Importantly, the levels of recombinant protein required are much less than that needed for other affinity techniques such as isothermal titration calorimetry and anisotropy fluorescence spectroscopy.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
1.Lavery, D.N. and McEwan, I.J. (2005) Structure and function of steroid receptor AF1 transactivation domains: induction of active conformations. Biochem.J., 391, 449–464.
2.Xu, J. and Li, Q. (2003) Review of the in vivo functions of the p160 steroid receptor coactiva-tor family. Mol.Endocrinol., 17, 1681–1692.
3.Treuter, E., Johansson, L., Thomsen, J.S., Warnmark, A., Leers, J., Pelto-Huikko, M., Sjoberg, M., Wright, A.P., Spyrou, G. and Gustafsson, J.A. (1999) Competition between thyroid hormone receptor-associated protein (TRAP) 220 and transcriptional intermediary factor (TIF) 2 for binding to nuclear receptors. Implications for the recruitment of TRAP and p160 coactivator complexes. J.Biol.Chem., 274, 6667–6677.
4.Burakov, D., Wong, C.W., Rachez, C., Cheskis, B.J. and Freedman, L.P. (2000) Functional interactions between the estrogen receptor and DRIP205, a subunit of the heteromeric DRIP coactivator complex. J.Biol.Chem., 275, 20928–20934.
5.Rich, R.L., Hoth, L.R., Geoghegan, K.F., Brown, T.A., LeMotte, P.K., Simons, S.P., Hens-ley, P. and Myszka, D.G. (2002) Kinetic analysis of estrogen receptor/ligand interactions. Proc. Natl.Acad.Sci.USA., 99, 8562–8567.
6.Warnmark, A., Wikstrom, A., Wright, A.P., Gustafsson, J.A. and Hard, T. (2001) The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties. J.Biol.Chem., 276, 45939–45944.
7.Shatkina, L., Mink, S., Rogatsch, H., Klocker, H., Langer, G., Nestl, A. and Cato, A.C. (2003) The cochaperone Bag-1L enhances androgen receptor action via interaction with the NH2-terminal region of the receptor. Mol.Cell.Biol., 23, 7189–7197.
8.Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal.Biochem., 72, 248–254.
9.Lavery, D.N. and McEwan, I.J. (2008) Functional characterization of the native NH2-terminal transactivation domain of the human androgen receptor: binding kinetics for interactions with TFIIF and SRC-1a. Biochemistry, 47, 3352–3359.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Lavery, D. (2009). Binding Affinity and Kinetic Analysis of Nuclear Receptor/Co-Regulator Interactions Using Surface Plasmon Resonance. In: McEwan, I.J. (eds) The Nuclear Receptor Superfamily. Methods in Molecular Biology™, vol 505. Humana Press. https://doi.org/10.1007/978-1-60327-575-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-60327-575-0_10
Publisher Name: Humana Press
Print ISBN: 978-1-60327-574-3
Online ISBN: 978-1-60327-575-0
eBook Packages: Springer Protocols