Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis

Sarcoplasmic reticulum Ca2+‐ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as α‐helix, β‐sheet, etc…, two unusual bands, one at 1,645 cm −1 in H2O buffer and the other at 1,625 cm−1 in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm−1 is tentatively assigned to α‐helical segments located in the cytoplasmic domain and coupled to β‐sheet structure, whereas the band at 1,625 cm−1 arises probably from monomer‐monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% α‐helical segments in the transmembrane plus stalk domain; 20% α‐helix and 22% β‐sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+‐ATPase is a complex process that cannot be described as a two‐state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm −1 band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm −1 band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 °C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.