Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

Marie-Elaine Caruso,Sarah Jenna,Simon Beaulne,Eun-Hye Lee,Anne Bergeron,Cédric Chauve,Philippe Roby,Jean-François Rual,David E Hill,Marc Vidal,Roger Bossé,Eric Chevet

doi:10.1074/mcp.m500025-mcp200

Abstract

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen and FlashPlate, to measure nucleotide binding capacity, exchange, and hydrolysis activities of small monomeric GTPases. These two technologies have the characteristics to be very sensitive and to allow homogenous and high throughput assays. To analyze and integrate the data obtained, we developed an algorithm that allows the classification of GTPases according to their enzymatic activities. Integration and hierarchical clustering of these results revealed unexpected features of the small Rho GTPases when compared with primary sequence-based trees. Hence we propose a novel phylobiochemical classification of the Ras superfamily of GTPases.

Highlights

To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence
Rho GTPases Identified in the C. elegans Genome—The ORFeome projects, which consist of the individual cloning of most protein-encoding ORFs from the ATG to the stop codon predicted from genome sequences, are the cornerstone of system biology approaches
The GST fusion proteins were expressed at high levels and purified to homogeneity (Fig. 1B). Using these small G proteins, we developed four distinct assays to measure their affinity for guanine nucleotides as well as their exchange and hydrolysis activities

Summary

Biochemical Clustering of Ras GTPases

In this study we took advantage of these two technologies to characterize in a systematic manner four biochemical properties of the Ras family of small G proteins and we reclassified them according to their activities instead of their primary amino acid sequences. Due to their high level of interspecies conservation, we selected the Rho GTPases as prototype proteins to develop and validate our assays. The plate was incubated for 20 min, and 25 nM goat anti-GST (Amersham Biosciences) was added. After 20 min of incubation, Donor and Acceptor beads were added simultaneously, and the plate was analyzed after 1 h of incubation at 23 °C

Plasmids and Constructs

Nucleotide Biotinylation

AlphaScreen Assays

FlashPlate Assays

RESULTS

DISCUSSION