Size Determination of Cyanobacterial and Higher Plant Photosystem II by Gel Permeation Chromatography, Light Scattering, and Ultracentrifugation
Size Determination of Cyanobacterial and Higher Plant Photosystem II by Gel Permeation Chromatography, Light Scattering, and Ultracentrifugation
Received October 29, 2004
Revised Manuscript Received December 21, 2004
Web Release Date: February 25, 2005
Athina Zouni,* Jan Kern, Joachim Frank, Thomas Hellweg, Joachim Behlke, Wolfram Saenger, and Klaus-Dieter Irrgang*
Biochemistry
ACS Publications
Copyright ? 2005 American Chemical Society
Max-Volmer-Laboratorium, Technische Universit?t Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany, Iwan-N.-Stranski-Laboratorium, Technische Universit?t Berlin, Strasse des 17. Juni 122, D-10623 Berlin, Germany, Max-Delbr?ck-Centrum f?r Molekulare Medizin Berlin Buch, Robert-R?ssle-Strasse 10, D-13092 Berlin, Germany, and Institut f?r Kristallographie, Freie Universit?t Berlin, Takustrasse 6, D-14195 Berlin, Germany
Abstract:
The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl--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 PSIIcc 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 al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 ?, which has been improved recently to 3.2 ? [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].
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