Ca²⁺ ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR

To transmit signals across cellular compartments, many membrane-embedded enzymes undergo extensive conformational rearrangements. Monitoring these events in lipid bilayers by NMR at atomic resolution has been challenging due to the large size of these systems. It is further exacerbated for large mammalian proteins that are difficult to express and label with NMR-active isotopes. Here, we synthesized and engineered ¹³C ethyl groups on native cysteines to map the structural transitions of the sarcoplasmic reticulum Ca²⁺-ATPase, a 110 kDa transmembrane enzyme that transports Ca²⁺ into the sarcoplasmic reticulum. Using magic angle spinning NMR, we monitored the chemical shifts of the methylene and methyl groups of the derivatized cysteine residues along the major steps of the enzymatic cycle. The methylene chemical shifts are sensitive to the ATPase conformational changes induced upon nucleotide and Ca²⁺ ion binding and are ideal probes for active and inactive states of the enzyme. This new approach is extendable to large mammalian enzymes and signaling proteins with native or engineered cysteine residues in their amino acid sequence.