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 alpha-helix, beta-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 alpha-helical segments located in the cytoplasmic domain and 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 segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-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 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.
Echabe
I
I
Grupo de Biomembranas, Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.
Dornberger
U
U
Prado
A
A
Goñi
F M
FM
Arrondo
J L
JL
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D006868:Hydrolysis; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013055:Spectrophotometry, Infrared
D000252:Calcium-Transporting ATPases