Size determination of cyanobacterial and higher plant photosystem II by gel permeation chromatography, light scattering, and ultracentrifugation.
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-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 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 A, which has been improved recently to 3.2 A [Biesiadka, J., et al. (2004) Phys. Chem. Chem. Phys. 6, 4733-4736].
Zouni
Athina
A
Max-Volmer-Laboratorium, Technische Universität Berlin, Strasse des 17. Juni 135, D-10623 Berlin, Germany. Zouni@phosis1.chem.tu-berlin.de
Kern
Jan
J
Frank
Joachim
J
Hellweg
Thomas
T
Behlke
Joachim
J
Saenger
Wolfram
W
Irrgang
Klaus-Dieter
KD
D001426:Bacterial Proteins / Q000737:chemistry*; D005591:Chemical Fractionation; D002850:Chromatography, Gel / Q000379:methods; D003460:Crystallization; D000458:Cyanobacteria; D003720:Densitometry; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry; D008956:Models, Chemical; D008970:Molecular Weight; D045332:Photosystem II Protein Complex / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D012542:Scattering, Radiation; D012996:Solutions; D013053:Spectrophotometry; D018724:Spinacia oleracea; D020524:Thylakoids / Q000737:chemistry; D014461:Ultracentrifugation / Q000379:methods
D001426:Bacterial Proteins; D008565:Membrane Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D012996:Solutions