Biochemical Assays to Characterize Rho GTPases
Rho GTPases act as tightly regulated molecular switches governing a large variety of critical cellular functions. Their activity is controlled by two different biochemical reactions, the GDP/GTP exchange and the GTP hydrolysis. These very slow reactions require catalysis in cells by two kinds of regulatory proteins. While the guanine nucleotide exchange factors (GEFs) activate small GTPases by stimulating the exchange of bound GDP for the cellular abundant GTP, GTPase-activating proteins (GAPs) accelerate the intrinsic rate of GTP hydrolysis by several orders of magnitude, leading to their inactivation. There are a number of methods that can be used to characterize the specificity and activity of such regulators to understand the effect of binding on the protein structure and, ultimately, to gain insights into their biological functions. This chapter describes (1) detailed protocols for the expression and purification of Rho GTPases, of effector-binding domains, and catalytic domains of GEFs and GAPs; (2) the preparation of nucleotide-free and fluorescent nucleotide-bound Rho GTPases; and (3) methods for monitoring the intrinsic and GEF-catalyzed nucleotide exchange, the intrinsic and GAP-stimulated GTP hydrolysis, and the effector interaction with active GTPase (three alternative approaches).
Key wordsFluorescence spectroscopy GAP GEF GTPase Guanine nucleotide Mant Protein–protein interactions Rho Tamra Effector
- 14.Eberth, A., and Ahmadian, M.R. (2009) In vitro GEF and GAP assays. In: Bonifacino, J.S, Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds) Current Protocols in Cell Biology, Unit 14.9. Wiley, New York.Google Scholar
- 24.Gremer, L., De Luca, A., Merbitz-Zahradnik, T., Dallapiccola, B., Morlot, S., Tartaglia, M., Kutsche, K., Ahmadian M.R., and Rosenberger, G. (2010) Duplication of Glu37 in the switch I region of HRAS impairs effector/GAP binding and underlies Costello syndrome by promoting enhanced growth factor-dependent MAPK and AKT activation Hum Mol Genet 19, 790–802.PubMedCrossRefGoogle Scholar