Crystallization of reaction center from Rhodopseudomonas sphaeroides: preliminary characterization.

Abstract

Reaction centers (RC), integral membrane proteins that mediate the conversion of light into chemical energy, were crystallized by 2 different vapor diffusion techniques. In 1 method, small amphipathic molecules (1,2,3-heptanetriol and triethylammonium phosphate) were added to the RC that had been solubilized in detergent. In the 2nd method, crystallization occurred near the phase boundaries of a 2-phase system created by the addition of polyethylene glycol and NaCl to RC in octyl .beta.-D-glucoside. Several different crystal forms were obtained; 2 were analyzed by X-ray diffraction. One was monoclinic (space group P2) with .beta. = 105.degree., and a = 70 .ANG., b = 105 .ANG., and c = 85 .ANG., 2 RC/unit cell, and 1 RC/asymmetric unit; the crystal diffracted to 3.5 .ANG. at 17.degree. C. The other crystal form was orthorhombic (space group C222) with a = 185 .ANG., b = 170 .ANG., and c = 105 .ANG., with 8 RC/unit cell and 1 RC/asymmetric unit. Reversible light-induced EPR signals of the primary donor (bacteriochlorophyll dimer) showed that the RC in the crystal were fully active. From the angular dependence of the EPR signal the molecular g [signal-splitting value] anisotropy of the bacteriochlorophyll dimer was deduced to be g.perp. - g.dblvert. = (64 .+-. 3) .times. 10-5. Linear dichroism measurements were performed on the monoclinic crystal. The 2 bands at 535 and 544 nm assigned to the Qx transitions of the bacteriopheophytins were resolved and preliminary orientations of some of the pigments were obtained.