In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases

Alyssa M Blaise; Ellen E Corcoran; Eve S Wattenberg; Yan-Ling Zhang; Jeffrey R Cottrell; Anthony J Koleske

doi:10.1093/biomethods/bpab024

In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases

Biol Methods Protoc. 2021 Dec 31;7(1):bpab024. doi: 10.1093/biomethods/bpab024. eCollection 2022.

Authors

Alyssa M Blaise^{1

2}, Ellen E Corcoran^{1

2}, Eve S Wattenberg^{1

2}, Yan-Ling Zhang³, Jeffrey R Cottrell³, Anthony J Koleske^{1

2}

Affiliations

¹ Departments of Molecular Biophysics and Biochemistry, Yale School of Medicine, Yale University, New Haven, CT 06520-8024, USA.
² Neuroscience, Yale School of Medicine, Yale University, New Haven, CT 06520-8024, USA.
³ Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Abstract

Guanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed, user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.

Keywords: Rho family GTPase; Trio protein; guanine nucleotide exchange factor (GEF).

Abstract

Grants and funding