Correlation between the conformational states of F 1 -ATPase as determined from its crystal structure and single-molecule rotation

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Abstract
F 1 -ATPase is a rotary molecular motor driven by ATP hydrolysis that rotates the γ-subunit against the α 3 β 3 ring. The crystal structures of F 1 , which provide the structural basis for the catalysis mechanism, have shown essentially 1 stable conformational state. In contrast, single-molecule studies have revealed that F 1 has 2 stable conformational states: ATP-binding dwell state and catalytic dwell state. Although structural and single-molecule studies are crucial for the understanding of the molecular mechanism of F 1 , it remains unclear as to which catalytic state the crystal structure represents. To address this issue, we introduced cysteine residues at βE391 and γR84 of F 1 from thermophilic Bacillus PS3. In the crystal structures of the mitochondrial F 1 , the corresponding residues in the ADP-bound β (β DP ) and γ were in direct contact. The βE190D mutation was additionally introduced into the β to slow ATP hydrolysis. By incorporating a single copy of the mutant β-subunit, the chimera F 1 , α 3 β 2 β(E190D/E391C)γ(R84C), was prepared. In single-molecule rotation assay, chimera F 1 showed a catalytic dwell pause in every turn because of the slowed ATP hydrolysis of β(E190D/E391C). When the mutant β and γ were cross-linked through a disulfide bond between βE391C and γR84C, F 1 paused the rotation at the catalytic dwell angle of β(E190D/E391C), indicating that the crystal structure represents the catalytic dwell state and that β DP is the catalytically active form. The former point was again confirmed in experiments where F 1 rotation was inhibited by adenosine-5′-(β,γ-imino)-triphosphate and/or azide, the most commonly used inhibitors for the crystallization of F 1 .

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