Development of TOXGFP Assay to Measure Transmembrane Interactions

Abstract

Association of transmembrane alpha-helices is a fundamental part of membrane protein structure. The development of an assay (TOXGFP) allows for the efficient study of transmembrane helix oligomerization in live cells. The N-terminal DNA binding domain ToxR is a dimerization-dependent transcriptional activator, which is fused to the transmembrane domain of interest. Association of the transmembrane domains, leads to the activation of the reporter gene encoding green fluorescent protein (GFP). The level of fluorescence indicates the amount of GFP expressed, which correlates with the strength of the transmembrane association. Using a known dimerizing transmembrane domairis and a mutant variant known for low dimerization, the sensitivity of the TOXGFP system is shown. The TOXGFP assay allows for the high throughput analysis of a large range of transmembrane domain oligomerization motifs.