Crystallization of membrane proteins: bovine rhodopsin.

E V Yurkova, V V Demin, N G Abdulaev

Index: Biomed. Sci. 1(6) , 585-90, (1990)

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Abstract

The conditions for crystallization of the integral membrane protein bovine rhodopsin were determined. Crystals form over a wide range of protein concentration (0.4-15 mg ml-1) and pH (5.5-7.5) in detergent solutions of octyl-polyoxyethylene (o-POE) that contain inorganic salts as precipitants. Crystallization occurs in the detergent-rich phase under conditions of phase separation. This corroborates the results of experiments on the phase behavior of the detergent solution and on the distribution of rhodopsin between the salt-rich and detergent-rich phases formed at high salt concentrations. Crystals were needle shaped, and the best crystals were obtained by vapour diffusion, at room temperature, of a 2.5 mg ml-1 protein solution in a 1.5% (v/v) o-POE solution containing 1.5 M ammonium sulphate (pH 7.0) against unbuffered 2.85 M ammonium sulphate. The crystals thus produced had dimensions of approximately 70 microns x 70 microns x 1000 microns. These crystals were too small to allow X-ray diffraction studies of rhodopsin structure, but electron microscopic studies of negatively stained thin crystals allowed the definition of the projection belonging to the two-sided plane group p21, and showed unit cell parameters alpha (50 A), b (72 A), and gamma (90 degrees).