A FlAsH-tetracysteine assay for quantifying the association and orientation of transmembrane α-helices.

The association of membrane proteins with a single transmembrane a-helix (TMH) has been shown to play a critical role in numerous cellular processes. For example, TMH dimerization of the ErbB family of receptor tyrosine kinases (RTKs) results in trans-phosphorylation and subsequent activation of the downstream pathways of growth factor signalling. A series of naturally occurring, single amino acid mutations in the TMH domain of these RTKs have been linked to a number of diseases, presumably because these mutations perturb TMH dimerization propensities. For example, the dimer promoting mutation V664E in the TMH domain of ErbB2 (HER2) has been associated with an increased risk of cancer. This physiological importance is reflected in the high number of drugs that target membrane proteins (ca. 50%) relative to their distribution in the proteome (ca. 30%). Although significant progress has been made in our mechanistic understanding of protein association in membranes, it remains difficult to predict the oligomerization state of a TMH and rationalize the (patho)physiological consequences of a mutation within a membrane protein. While the association of soluble proteins is primarily driven by the hydrophobic effect, protein–protein interaction within membranes is thought to rely on weak molecular interactions such as sidechain hydrogen bonding and van der Waal’s packing. In order to gain a better understanding of these energetic factors and ultimately develop novel inhibitors of TMH association, a simple and efficient assay is highly desirable for quantifying TMH association both in vitro (detergent micelles and liposomes) and in living cells. Biarsenical dyes, such as 4’,5’bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH), are an intriguing tool for monitoring protein–protein interaction in membranes. In their EDT (ethane-1,2-dithiol)-protected forms, these dyes (e.g. , FlAsH–EDT2) are essentially nonfluorescent. However, thiol–arsenic ligand exchange with a tC motif elicits a strong fluorescence emission. These fluorophores have been applied to a number of soluble proteins to report their folding and stability, and even subtle conformational changes. A recent development in this area has resulted in a technique known as “bipartite tC display”, in which the biarsenical dyes are used to monitor the dimerization of water-soluble proteins. Herein, we describe the development of a FlAsH–tC assay for quantifying the association of membrane-embedded proteins. While the commonly used methods, such as SDSPAGE and analytical ultracentrifugation (AUC) are limited to micellar systems, the FlAsH–tC assay allows facile evaluation of protein dimerization in both micelles and lipid bilayers. Importantly, the FlAsH fluorescence reports TMH dimerization in an orientation-specific manner (vide infra), which is advantageous over the previously reported, FRET-based assays. As a model system, we first synthesised a pair of polyleucine (pL)-based peptides (Figure 1B). These peptides were chosen because their oligomerization state had been previously characterized in both micelles (by AUC and SDS-PAGE) and the