Abstract
Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F1-ATPase, mostly based on F1 from thermophilic bacteria. In contrast, high-resolution crystal structures are only available for mitochondrial F1. Here we present high resolution single molecule rotational data on F1 from Saccharomyces cerevisiae, obtained using new high throughput detection and analysis tools. Rotational data are presented for the wild type mitochondrial enzyme, a “liver” isoform, and six mutant forms of yeast F1 that have previously been demonstrated to be less efficient or partially uncoupled. The wild-type and “liver” isoforms show the same qualitative features as F1 from Escherichia coli and thermophilic bacteria. The analysis of the mutant forms revealed a delay at the catalytic dwell and associated decrease in Vmax, with magnitudes consistent with the level of disruption seen in the crystal structures. At least one of the mutant forms shows a previously un-observed dwell at the ATP binding angle, potentially attributable to slowed release of ADP. We discuss the correlation between crystal structures and single molecule results.
Highlights
Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F1-ATPase, mostly based on F1 from thermophilic bacteria
The analysis of the mutant forms revealed a delay at the catalytic dwell and associated decrease in Vmax, with magnitudes consistent with the level of disruption seen in the crystal structures
TP, E and DP states are best defined by their position with respect to the asymmetric c subunit, as their nucleotide occupancy varies across different crystal structures
Summary
Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F1-ATPase, mostly based on F1 from thermophilic bacteria. A mechanochemical cycle can be defined whereby a single b subunit in the E* conformation binds ATP at 0u, moves through the TP and TP* conformations to hydrolyse ATP in the DP conformation at 200u and release ADP at 240u (DP*). TF1 and EF1 rotate unidirectionally but not continuously, stochastically entering a paused state due to Mg21 ADP inhibition[13]
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