Analyzing Transmembrane Protein and Hydrophobic Helix Topography by Dual Fluorescence Quenching

The location of fluorescent groups relative to the lipid bilayer can be evaluated using fluorescence quenchers embedded in the membrane and/or dissolved in aqueous solution. Quenching can be used to define the membrane topography of membrane proteins and individual membrane-embedded hydrophobic helices by combining it with the placement of fluorescent groups, including Trp, at defined sequence positions. This chapter briefly discusses various quenching methods for studies of membrane protein topography and provides detailed protocols for dual quencher analysis (DQA), a rapid, highly sensitive, and experimentally flexible approach in which the information gained from both a membrane-embedded and aqueous quencher is combined. The advantages of the DQA method include flexibility with regard to the bilayer compositions to which it can be applied, including membranes composed of lipids of varying headgroup and acyl chain compositions, as well as the ability to identify mixed populations of fluorophores residing at different depths within the bilayer.