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

Cell membranes function as highly efficient biological barriers, separating the cytosol from the extracellular space. Membrane transporters are membrane-embedded proteins that catalyze the translocation of ions and small molecules across cell membranes. Due to the hydrophobic environment within the cell membrane, membrane transporters have developed structures and mechanisms distinct from those of water-soluble proteins, making them much more challenging to characterize. Over the past several decades, extensive efforts have been devoted to developing experimental approaches for elucidating the structural and mechanical properties of these transport proteins. One of the powerful and widely adopted methods is the Substituted Cysteine Accessibility Method (SCAM). By exploiting the chemical versatility of the thiol group in cysteine, SCAM strategically replaces selected amino acids in a membrane transporter of interest with cysteines and assesses the accessibility of the introduced thiol groups using biochemical or biophysical probes under different experimental conditions, thereby providing valuable insights into the transmembrane topology, three-dimensional structure, conformational changes and transport mechanism of the transporter. The over three decades-long SCAM studies on the lactose transporter of Escherichia coli (LacY) represent a classic example of the application of SCAM to membrane transporters and have profoundly influenced the field of transporter biology. With a focus on SCAM investigations of LacY, this review article summarizes the applications of SCAM, discusses recent advancements, and suggests future directions.