Single-molecule biophysics of ATP synthase

Single-molecule biophysics of
ATP synthase

Single-molecule biophysics of ATP synthase has been one of the major interests of our lab. ATP synthase is a complex of two rotary molecular motors, F1 and Fo. Since the development of the rotary assay of F1 in 1997 [1], we have been studying chemo-mechanical coupling and regulatory mechanisms of ATP synthase by analyzing its conformational and reaction dynamics at single-molecule level [2].
We have revealed most of the chemo-mechanical coupling reaction scheme of F1. However, the timing of phosphate release is yet to be deciphered. This is crucial specially for understanding the force-generation mechanism of F1. One of our current approaches toward this is the structure analysis of F1 crystalized with bound phosphate or phosphate analog. We are also keen to develop novel methods for the phosphate release detection from a single F1 molecule.
Another question that interests our lab is how the whole complex of ATP synthase (FoF1) rotates coupling proton translocation and ATP hydrolysis/synthesis. The technical challenges that have prevented the single-molecule analysis of ATP synthase till date not only include the difficulty of its purification and reconstitution into suitable membrane systems, but also that of applying sufficiently large membrane potential across the ATP synthase-reconstituted membrane. For this purpose, we had previously developed a supported membrane system that allows us to charge transient membrane potential to drive the rotation of the whole complex of ATP synthase [3]. To enhance the stability of membrane integrity as well as membrane potential, we developed arrayed lipid bilayer chamber systems (ALBiCs), and achieved single-molecule analysis of active proton transport by ATP synthase [4]. 1. H. Noji et al., 1997, Nature 386, 299-302
2. H. Noji, H. Ueno, and D.G.G. Duncan, 2016 Biophys. Rev. 9, 103-118
3. R. Watanabe et al., 2013 Nat. Commun. 4, 1631
4. R. Watanabe and N. Soga et al., 2014 Nat. Commun. 5, 4519

野地研究室 Single Molecule Biophysics Noji Laboratory