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

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Abstract
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.