Comparison between single-molecule and X-ray crystallography data on yeast F 1-ATPase

Abstract

Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F 1-ATPase, mostly based on F 1 from thermophilic bacteria. In contrast, high-resolution crystal structures are only available for mitochondrial F 1. Here we present high resolution single molecule rotational data on F 1 from Saccharomyces cerevisiae, obtained using new high throughput detection and analysis tools. Rotational data are presented for the wild type mitochondrial enzyme, a isoform, and six mutant forms of yeast F 1 that have previously been demonstrated to be less efficient or partially uncoupled. The wild-type and isoforms show the same qualitative features as F 1 from Escherichia coli and thermophilic bacteria. The analysis of the mutant forms revealed a delay at the catalytic dwell and associated decrease in V max, 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.