Protein Science, Vol 7, Issue 5 1172-1179, Copyright © 1998 by Cold Spring Harbor Laboratory Press

ARTICLE

Topology of sarcoplasmic reticulum Ca(2+)-ATPase: An infrared study of thermal denaturation and limited proteolysis

I. ECHABE, U. DORNBERGER, A. PRADO, F. M. GONI and JLR. ARRONDO
Grupo de Biomembranas, Departamento de Bioquimica, Universidad del Pais Vasco, P.O. Box 644, E-48080 Bilbao, Spain

Sarcoplasmic reticulum Ca(2+)-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 H(2)O buffer and the other at 1,625 cm(-1) in D(2)O 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 Ca(2+)-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 Ca(2+) accumulation and ATPase activity are uncoupled at temperatures above 37{deg}C, so that while ATPase activity proceeds at high rates, Ca(2+) accumulation is inhibited.
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