Substituted cysteine accessibility method (SCAM) in membrane transporters studies: Learn from lactose permease

Membrane transporters are membrane-embedded proteins that mediate the translocation of a great variety of ions and small molecules across biological membranes. Due to the highly hydrophobic environment in which they reside, transporters have evolved structures and mechanisms distinct from those of soluble cytoplasmic proteins, making them considerably more challenging to solubilize, purify, and characterize. As a result, our current understanding of membrane transporters remains far more limited than that of soluble proteins. Since its emergence in the early 1990s, Substituted Cysteine Accessibility Method (SCAM) has become a powerful and widely adopted approach for the structure-function studies of membrane proteins, including transporters, channels and receptors. By exploiting the chemical versatility of the cysteine thiol group, SCAM, when combined with appropriate biochemical and biophysical techniques, provides valuable insights into transmembrane topology, three-dimensional structure, conformational dynamics, transport mechanisms, and protein-protein or lipid-protein interactions. The over three decades-long SCAM studies on the lactose permease of Escherichia coli (LacY), a paradigm of the Major Facilitator Superfamily (MFS), represent a classic example of the application of SCAM to transporters and have profoundly influenced the field of transporter biology. In this review, we focus on SCAM investigations of LacY, summarize the broader applications of SCAM to membrane transporters, discuss recent advancements, and suggest future directions.