Analysis of GEF-Mediated Nucleotide Exchange Assay Experiments

• Activity Number: 423
• Type: Contributed
• Date/Time: Tuesday, August 2, 2011 : 2:00 PM to 3:50 PM
• Sponsor: Biopharmaceutical Section
• Author(s): Qinghua Song*+ and Lindsay Garrenton and Imola Fodor and Guowei Fang and Peter Jackson
• Companies: Genentech Inc. and Genentech Inc. and Genentech Inc. and Genentech Inc. and Genentech Inc.
• Keywords: GEF ; exponential decay model ; four-parameter logistic fit

Abstract

Guanine nucleotide exchange factors (GEF) are components of intracellular signaling networks that activate downstream proteins by stimulating the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP). Son of Sevenless (SOS) is a Ras-specific GEF that activates Ras proteins, which play a role in regulating cell growth, differentiation, and survival, and have been implicated in many types of cancer. To biochemically characterize GEF-mediated nucleotide exchange, we developed fluorescence-based GEF assays and statistical methods for their analysis. We fit an exponential decay model to the time series of each enzyme concentration and estimate the corresponding exponential decay rates. We then display the ratio of estimated rates versus that of no enzyme control as a function of increasing log enzyme concentration and estimate the EC50 for the exchange reaction via a four-parameter logistic fit. We illustrate the data analysis steps and results with an assay for a titration of the catalytic subunit of the RasGEF, SOS1. The automated data analysis process we developed will facilitate the interpretation of multiple future experiments.