Two‐dimensional crystallization of reaction centers from Chloroflexus aurantiacus

Abstract

Two-dimensional crystals of photosynthetic reaction centers from CMarqkus auranfiacus were obtained from protein-lipid-detergent micelles by detergent dialysis. The size of crystals was up to 2 e. Some of them were multi~yer~ crystals. However, other crystal forms were also observed. Preliminary image processing analysis showed that crystals of one crystal form referred to two-sided piane group p2 and had the following unit cell parameters: a= 17,6 nm, b= I8,O nm, y=84". The contour map of the crystal stain-excluding region was calculated by the Fourier-filtering procedure at about 2 nm resolution.Reaction center; Two~imensionai crystal; Electron microscopy; Image processing 1" INTRODUCTIONPrimary photochemical reactions in bacterial photosynthesis are known to occur in integral membrane protein systems, i.e. reaction centers (RC). Although the detailed mechanisms of the functioning of these muitimolecular systems are still obscure, the studies of the structural organization of the photosynthetic systems at all levels are important. The membrane organization of the bacteria RC is under extensive study. The structure of the RC molecules of purple bacteria Rhodopseudoareas viridis [ 1,2] and R~odops~d~~onus sphaeroids [3,4] were determined at atomic resolution by X-ray analysis. The spatial structure of the photosynthetic RC of green bacteria C~loro~ex~s aurant~ac~s in the membrane is as yet unknown. Staehelin with coworkers analyzed a general morphology of Cklorofrexus aurantiacus bacteria 151. Recently primary structures of the L-and ~-subunits of C~~oro~~s RC were established, that allowed modeling the topography of the polypeptide chains in the membrane [S, 71.The structural organization of membrane proteins can be studied by electron microscopy of two-dimensional crystals followed by computer image reconstruction techniques [8], The present work was aimed at twodimensional crystallization of Ckloroflexus RC and 2, MATERIALS AND METHODSIsolation and purification of RC, determination of spectral and other chemical parameters were carried out as described in [4,5]. Lecithin from soybean (Sigma) was solubilized by IauryI~methyl-amine N-oxide (LDAO). For reconstitution experiments, solubilized lecithin was added to the RC sample (10 mg/ml of protein in 50 mM of Tris-HCl, pH 9.0, &l@?o LDAO) at various proportions. Then detergent was removed by dialysis under various conditions (temperature, dialysis rate, ionic strength, ion composition of dialysis media) 181.Electron microscopy studies, optical diffraction and image processing were done as described in [IO].

Two-dimensional crystallization of membrane proteins

“…Dialysis of bacterial photosynthetic reaction centre from R. viridis solubilized in LDAO produced 2D crystals measuring more than 1 /im in diameter (Miller & Jacob, 1983). Even though these 2D crystals looked promising, they were not used for high-resolution data collection because the structure of the reaction centre was already being determined by X-ray crystallography of 3D crystals (Deisenhofer et al 1984(Deisenhofer et al , 1985)-2D crystals of reaction centre complex from the green bacterium Chloroflexus aurantiacus solubilized in LDAO were obtained by reconstitution with soybean PC (Barnakov et al 1990). Judging by the projected structure at low resolution, the crystal packing seems to be similar to that in 2D crystals of the R. viridis reaction centre (Miller & Jacob, 1983; see §3.3.1 e).…”