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

• Published in: The Journal of biological chemistry 1988-07, Vol.263 (19), p.9162-9170
• Main Authors: Squier, T C, Hughes, S E, Thomas, D D
• Format: Article
• Language: English
• Subjects: adenosinetriphosphatase; Animals; Biological and medical sciences; Calcium-Transporting ATPases - metabolism; Conformational dynamics in molecular biology; Cross-Linking Reagents - pharmacology; Cyclic N-Oxides - pharmacology; Electron Spin Resonance Spectroscopy; Fluorescein-5-isothiocyanate; Fluoresceins - pharmacology; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Kinetics; membrane proteins; Molecular biophysics; Muscles - enzymology; Protein Conformation; Rabbits; Rotation; sarcoplasmic reticulum; Sarcoplasmic Reticulum - enzymology; Succinimides - pharmacology; Thiocyanates - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)76521-1

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.