On the stator of rotary ATP synthase: the binding strength of subunit delta to (alpha beta)3 as determined by fluorescence correlation spectroscopy.
ATP synthase is conceived as a rotary enzyme. Proton flow drives the rotor (namely, subunits c12 epsilon gamma) relative to the stator (namely, subunits ab2 delta(alpha beta)3) and extrudes spontaneously formed ATP from three symmetrically arranged binding sites on (alpha beta)3 into the solution. We asked whether the binding of subunit delta to (alpha beta)3 is of sufficient strength to hold against the elastic strain, which is generated during the operation of this enzyme. According to current estimates, the elastically stored energy is about 50 kJ/mol. Subunit delta was specifically labeled without impairing its function. Its association with solubilized (alpha beta)3 gamma in detergent-free buffer was studied by fluorescence correlation spectroscopy (FCS). A very strong tendency of delta to dimerize in detergent-free buffer was apparent (K(d) </= 0.2 nM). Taking the upper limit of this figure into account, the dissociation constant between monomeric delta and (alpha beta)3 gamma was 0.8 nM if not smaller. It is equivalent to a free energy of binding of at least 52 kJ/mol and therewith is sufficient for the assumed hold-function of delta in the stator. Our data were compatible with a single binding site for delta on the hexagon of (alpha beta)3.
Häsler
K
K
Department of Biology/Chemistry, Division of Biophysics, Universität Osnabrück, Germany.
Pänke
O
O
Junge
W
W
D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D005227:Fatty Acids / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005944:Glucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D018892:Proton-Motive Force; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry; D012996:Solutions; D013050:Spectrometry, Fluorescence / Q000379:methods; D018724:Spinacia oleracea / Q000201:enzymology; D014783:Viscosity
D003902:Detergents; D005227:Fatty Acids; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D012235:Rhodamines; D012996:Solutions; C005358:tetramethylrhodamine; C042222:nonanoyl-N-methylglucamide; D006180:Proton-Translocating ATPases; D005944:Glucosamine