Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation

Zouni A, Kern J, Frank J, et al. Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation. Biochemistry. 2005;44(11):4572-4581.The oxygen-evolving photosystern 11 core complexes (PS11cc) from the thermophilic cyanobacteriurn Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl-beta-D-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC. DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PS11cc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 +/- 18 kDa and 710 +/- 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et a]. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 angstrom, which has been improved recently to 3.2 angstrom [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736]