Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies.

Abstract Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca 2+ -Mg 2+ )-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca 2+ -Mg 2+ )-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E 1 and E 2 states. The E 1 conformation was stabilized by 10 μM free Ca 2+ , while the E 2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid (EGTA). It was found that the E 1 and E 2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated α-helix content, respectively). In the presence of Ca 2+ , PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca 2+ -Mg 2+ )-ATPase by acrylamide, performed in the presence of Ca 2+ , gave evidence for a single class of tryptophan residues with Stern-Volmer constant ( K SV ) of 10 M −1 . Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the K SV value to 7.6 M −1 and 8.5 M −1 , respectively. In the absence of Ca 2+ , K SV was 7.0 M −1 . KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca 2+ -Mg 2+ )-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca 2+ . It is concluded that the interaction of CaM and PS with (Ca 2+ -Mg 2+ )-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E 1 and E 2 conformational forms of the ATPase.