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Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus
Author(s) -
Pogoryelov Denys,
Nikolaev Yaroslav,
Schlattner Uwe,
Pervushin Konstantin,
Dimroth Peter,
Meier Thomas
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06623.x
Subject(s) - protein subunit , atp synthase , chemistry , surface plasmon resonance , stereochemistry , biophysics , crystallography , rotor (electric) , dissociation (chemistry) , biochemistry , enzyme , biology , materials science , physics , nanotechnology , gene , quantum mechanics , nanoparticle
The interaction between the c 11 ring and the γε complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c 11 ring and the γε complex, with an apparent K d value of approximately 7.4 n m . The rotor assembly was primarily dependent on the interaction of the c ring with the γ subunit, and binding of the c ring to the free ε subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, γE204, γF203 and εH38) severely affected rotor assembly. The interaction kinetics between the γε complex and c 11 ring mutants suggested that the assembly of the c 11 γε complex was governed by interactions of low and high affinity. Low‐affinity binding was observed between the polar loops of the c ring subunits and the bottom part of the γ subunit. High‐affinity interactions, involving the two residues γE204 and εH38, stabilized the holo‐c 11 γε complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C‐ and N‐terminal regions of the γ subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F 1 complex to the rotor ring of F o to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F 1 F o complexes.