Rotational dynamics and protein-protein interactions in the Ca-ATPase mechanism.
We have varied the degree of protein-protein interactions among Ca-ATPase polypeptide chains in sarcoplasmic reticulum using the cleavable homobifunctional cross-linker dithiobissuccinimidyl propionate 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 specifically 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 (tau r), thus decreasing its rotational mobility (tau r-1). As the degree of cross-linking is varied, tau 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.
Squier
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TC
Department of Biochemistry, University of Minnesota Medical School, Minneapolis 55455.
Hughes
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Thomas
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D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011817:Rabbits; D012399:Rotation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013388:Succinimides / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
D003432:Cross-Linking Reagents; D003497:Cyclic N-Oxides; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides; D013861:Thiocyanates; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl; D000252:Calcium-Transporting ATPases; C011240:dithiobis(succinimidylpropionate); D016650:Fluorescein-5-isothiocyanate