Rotational dynamics and protein-protein interactions in the Ca-ATPase mechanism

T. C. Squier, S. E. Hughes, David D Thomas

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We have varied the degree of protein-protein interactions among Ca-ATPase polypeptide chains in sarcoplasmic reticulum using the cleavable homobifunctional cross-linker dithiobissuccinimidyl proprionate and have measured both the rotational mobility and calcium-dependent ATPase activity of the Ca-ATPase in order to assess 1) the nature of the microsecond rotational motion measured by saturation transfer EPR (ST-EPR) of the spin-labeled Ca-ATPase and 2) the functional significance of this rotational motion. The Ca-ATPase was labeled specificially and covalently with a maleimide spin label, with full preservation of enzymatic activity. ST-EPR experiments show that cross-linking increases the enzyme's effective rotational correlation time (τ(r), thus decreasing its rotational mobility (τ(r)-1). As the degree of cross-linking is varied, τ(r) is proportional to the mean molecular weight of the cross-linked aggregate, as predicted by theory, adding to the evidence that ST-EPR measures the overall rotational mobility of the Ca-ATPase with respect to the membrane normal. Furthermore, enzymatic activity correlates with overall protein rotational mobility, suggesting that this motion is functionally important. The second-order inactivation profile resulting from the use of either cross-linking or chemical modification with fluorescein isothiocyanate as modes of inactivation indicates that protein-protein interactions are critical to the reaction mechanism. However, the pattern of cross-linking observed on polyacrylamide gels demonstrates that cross-linking occurs in a random manner, indicating that no specific and stable oligomeric complexes exist. In order to rationalize both the functional need for protein mobility and the evidence that protein-protein interactions are critical and random, we propose that the enzymatic cycle of the Ca-ATPase involves the making and breaking of functionally important protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)9162-9170
Number of pages9
JournalJournal of Biological Chemistry
Issue number19
StatePublished - 1988


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