Work

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

Year: 1988

Type: article

Abstract: 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.

Source: Journal of Biological Chemistry

DOI: 10.1016/s0021-9258(19)76521-1

Authors: T C Squier, S E Hughes, D D Thomas

Institution: University of Minnesota

Language: English

Topic: ATP Synthase and ATPases Research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,430

Funders: National Institutes of Health, National Institute of General Medical Sciences