Specific heterodimer formation by the cytoplasmic domains of the b and b' subunits of cyanobacterial ATP synthase.
The soluble domains of the b and b' subunits of the ATP synthase of the cyanobacterium Synechocystis PCC 6803 were expressed with His tags attached to their N-termini. Following purification, the polypeptides were characterized by chemical cross-linking, analytical ultracentrifugation, and circular dichroism spectroscopy. Treatment of a mixture of the soluble b and b' domains with a chemical cross-linking agent led to substantial formation of cross-linked dimers, whereas similar treatment of either domain by itself resulted in only trace formation of cross-linked species. The molecular weights of the domains of b and b' in solution at 20 degrees C, measured by sedimentation equilibrium, were 17 800+/-700 and 16 300+/-400, respectively, compared to calculated polypeptide molecular weights of 16 635 and 15 422, whereas a mixture of b and b' gave a molecular weight of 29 800+/-800. The sedimentation coefficient of an equimolar mixture was 1.73+/-0.03. The circular dichroism spectra of the individual polypeptides indicated helical contents in the range of 40-50%; the spectrum of the mixture revealed changes indicative of coiled-coil formation and a helical content of 60%. The results indicate that the cytosolic domains of the b and b' subunits exist individually as monomers but form a highly extended heterodimer when they are mixed together.
Dunn
S D
SD
Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1. sdunn@julian.uwo.ca
Kellner
E
E
Lill
H
H
D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D000458:Cyanobacteria / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D019281:Dimerization; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012996:Solutions; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D012996:Solutions; D013388:Succinimides; C035760:bis(sulfosuccinimidyl)suberate; D006180:Proton-Translocating ATPases