TOPOLOGY OF SARCOPLASMIC-RETICULUM CA2-ATPASE - AN INFRARED STUDY OF THERMAL-DENATURATION AND LIMITED PROTEOLYSIS()

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the m embrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusu al 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 tempera ture and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain r ind coupled to beta-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% alpha-helical segmen ts in the transmembrane plus stalk domain; 20% alpha-helix and 22% bet a-sheet in the cytoplasmic domain plus 19% turns and 6% unordered stru cture. Thermal unfolding of Ca2+-ATPase is a complex process that cann ot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room tempe rature. The loss of the 1,625 cm(-1) band upon heating would be consis tent 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 temp eratures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.