Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology

Integral membrane proteins of the plasma membrane span from the inside to the outside of the cell. The primary structural element of integral membrane proteins is their topology: the pattern in which the protein traverses the membrane. A full description of topology, defining which parts of the protein face outside versus inside, goes a long way toward understanding the folding of these proteins. Many approaches have been established to define membrane protein topology. Here, we present the technique of scanning cysteine accessibility mutagenesis (SCAM). This approach uses the unique chemical reactivity of the cysteine sulfhydryl to probe membrane protein structure. Individual cysteine residues are introduced into the target protein by mutagenesis. The ability to chemically react these residues using sulfhydryl-directed reagents (either membrane permeant or impermeant) defines each site as either extracellular or intracellular, thus establishing topology of a location. This analysis performed on many sites in the protein will define the protein’s topology.