CRYSTALLIZATION OF A CALCIUM PUMP

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The Ca-ATPase of fast twitch muscle sarcoplasmic reticulum is representative of a class of ion pumps involved in the homeostasis of the cations Na+, K+, H+ and Ca+2. The similarities in structures and kinetics of these ion pumps (Na/K-ATPase, H/K- ATPase and Ca-ATPase) have been interpreted to indicate that they have been derived from a common ancestral gene (1). Ca+2 is unique among these cations in that if functions as a messenger in response to hormones and transmitters. Loss of Ca-pump activity is associated with such diseases as Duchenne muscular dystrophy (2), Brody's disease (3) and experimental myotonia (4). The work proposed here has the specific aim of crystallizing rabbit sarcoplasmic reticulum Ca-ATPase with the long-term objective of obtaining detailed structural information through application of diffraction methods to such crystals. Ca-ATPase will be purified in the laboratory of Prof. F.N. Briggs using procedures developed in that laboratory (5), which yield homogeneous enzyme. This material is solubilized in the presence of detergents, and will be used in this form as the starting material for crystallization trials. Ca-ATPase exists in two stable conformational forms, and in different states of aggregation. For crystallization, those conditions known to favor the monomer will be used. Trails to optimize these conditions will be made by combining and exchanging detergents, as was done in the case of porin crystallization. Aggregation state will be monitored either by analytical HPLC or centrifugation in the presence of detergent. Mild succinylation and amidination of the Ca-ATPase will also be tried, since the former has been shown to reduce aggregation. The two conformational states: high (E1) and low (E2) calcium affinity, will be selected through binding of ligands known to stabilize one of the two states. The more stable, Ca-bound E1 form will be screened for crystallization first. Divalent transition metals and several different nucleotides will be tested for their effects on crystallization. The E2 state exists as a dimer in vesicles and is stabilized by vanadate. Vanadate- bound enzyme will be screened in crystallization trials, varying detergent, precipitant concentrations and pH. Crystallization trials will be done by the sitting drop or microdialysis methods. Since Ca-ATPase has roughly equal extent of cytoplasmically exposed and intrinsic membrane regions, small amphiphilic molecules such as the heptane-triols will be tested for their effects in promoting crystallization. Any crystals obtained by these methods will be surveyed for lattice order by x-ray diffraction still or precession photographs.