Chloroplast coupling factor 1: dependence of rotational correlation time on polypeptide composition

Abstract

Time-resolved fluorescence depolarization measurements were made on chloroplast coupling factor 1 (CF1) labeled with pyrenylmaleimide. Rotational correlation times were determined for native CF1, for CF1 lacking epsilon and/or delta polypeptides, and for activated enzyme. The rotational correlation time measured is characteristic of the rotation of the entire enzyme. Removal of the delta polypeptide resulted in a 25% smaller rotational correlation time, although the delta polypeptide contributes less than 5% of the mass of CF1. Removal of the epsilon polypeptide was without effect. Simultaneous removal of delta and epsilon polypeptides produced a 30% smaller rotational correlation time. Activation of CF1 ATPase by incubation with dithiothreitol reduced the rotational correlation time by 15% relative to that of the latent enzyme. The rotational correlation time of CF1 with delta and epsilon polypeptides removed is essentially that expected for a spherical molecule, whereas the other forms of the enzyme can be approximated as ellipsoids of revolution; the axial ratio of the latent enzyme is estimated from the rotational correlation time and the intrinsic viscosity. These data indicate that the delta polypeptide significantly alters the shape of the enzyme and that a conformational change accompanies dithiothreitol activation of the enzyme.

Membrane-protein structural mapping of chloroplast coupling factor in asolectin vesicles

“…Similar results have been found for other fluorescent labels on this enzyme [cf. Hammes (1984, 1985)], and rates of rotation predominantly characteristic of the motion of the entire molecule have been found with dynamic fluorescence (Schinkel & Hammes, 1986) and phosphorescence (Musier-Forsyth & Hammes, 1990) measurements.…”

Field dependence of solvent proton and deuteron NMR relaxation rates of the manganese(II) binding site of chloroplast coupling factor 1

“…has been measured directly (Schinkel & Hammes, 1986), but this parameter appears only as a small correction (<3%) in the sum (l/rm + 1 /rsl + 1 /rr) and is of little significance. rd was estimated roughly from the fact that ts1 is already in the field-dependent region on the low-field side of the local maximum in 7?,, which implies that rd > 50 ps.…”

Characterization of three-subunit chloroplast coupling factor

“…CF,H,e) is a structurally simpler enzyme for study than CF,. Also, in contrast to the highly asymmetric shape of CF,, caused by the -polypeptide, CF,(-d,e) is approximately spherical (Schinkel & Hammes, 1986). It might, therefore, be possible to obtain crystals of CF,(-6,e) suitable for X-ray crystallography.…”

“…The structure of CFt has been extensively investigated with fluorescent methods (Cerione & Hammes, 1982;Cerione et al, 1983;Snyder & Hammes, 1984; Nalin et al, 1985;Richter et al, 1985;Schinkel & Hammes, 1986; McCarty & . Studies of nucleotide binding sites have revealed the existence of a site that binds ADP tightly (site 1), a site that binds MgATP tightly (site 2), and a site that binds ADP and ATP with a dissociation constant in the micromolar range (site 3) (Bruist & Hammes, 1981.…”

“…0006-2960/88/0427-0245S01.50/0 of catalysis and probably in proton translocation (McCarty & Moroney, 1985). The -polypeptide has no effect on the ATPase activity of CFb but it makes an important contribution to the asymmetric shape of CFt (Schinkel & Hammes, 1986) and to the efficient coupling of catalysis and proton pumping. The e-polypeptide is an ATPase inhibitor (Richter et al, 1985).…”