Crystallization and Linear Dichroism Measurements of the B800-850 Antenna Pigment-Protein Complex from Rhodopseudomonas sphaeroides 2.4.1

Abstract

Crude chromatophore membranes of R. sphaeroides 2.4.1 were purified by differential centrifugation and suspended in buffer (20 mM Tris-HCl, pH 8.0, 2mM Tris — EDTA) to a final absorbance A ⁸⁵⁰ _1cm. An equal volume of 1.6% (w/v) β;-D-octylglucopyranoside (βOG) in water was added at room temperature, and the resulting solubilizate centrifuged at ~2×10⁵ g for 60 min. at 4° C. The supernatant was brought to room temperature, and 10% nonanoyl-N-methyl-glucamide (Nga) was added to a final concentration of 1%. The B800-850-complex was isolated by column chromatography on DEAE-cellulose in the presence of 1% Nga and buffer (10 mM Tris-HCl, pH 8.0, lmM EDTA-Tris, 1 mM β;-mercaptoethanol). The B800-850-complex was eluted with 200 mM NaCl in Nga and buffer. Further purification was accomplished by repeating the DEAE chromatography. For the crystallization, 1% βOG was substituted for Nga by repeated concentration and dilution in an Amicon concentration cell and a final dialysis.

Work supported by the National Science Foundation (GM-13191) and the National Institute of Health (PCM 82-02811-AO3).