Mapping of Membrane Protein Topology by Substituted Cysteine Accessibility Method (SCAM™)

A described simple and advanced protocol for the substituted-cysteine accessibility method as applied to transmembrane (TM) orientation (SCAM™) permits a topology analysis of proteins in their native state and can be universally adapted to any membrane system to either systematically map an uniform topology or identify and quantify the degree of mixed topology. In this approach, noncritical individual amino acids that are thought to reside in the putative extracellular or intracellular loops of a membrane protein are replaced one at a time by cysteine residue, and the orientation with respect to the membrane is evaluated using a pair of membrane-impermeable nondetectable and detectable thiol-reactive labeling reagents.