“Breaking Speed and Resolution Limitations of High-Speed AFM for Membrane Protein Structure-Function Analysis”

Simon Scheuring1,2,*
1 Weill Cornell Medicine, Department of Anesthesiology, 1300 York Avenue, New York, NY-10065, USA.
2 Weill Cornell Medicine, Department of Physiology and Biophysics, 1300 York Avenue, New York, NY-10065, USA

High-speed atomic force microscopy (HS-AFM) is a powerful technique that provides dynamic movies of biomolecules at work [1]. We successfully used HS-AFM to take movies and determine dynamic parameters of membrane trafficking systems, transporters and channels.

To break current temporal limitations to characterize molecular dynamics using HS-AFM, we developed HS-AFM line scanning (HS-AFM-LS) and HS-AFM height spectroscopy (HS-AFM-HS), methods whereby we scan the HS-AFM tip along a single scan line or keep it at a fixed position, respectively, and detect the motions of the molecules under the tip. This gives sub-nanometer spatial resolution combined with millisecond and microseconds temporal resolution of molecular fluctuations. HS-AFM-LS and HS-AFM-HS can be used in conjunction with HS-AFM imaging, thus giving access to a wide dynamic range [1]. This allowed us most recently to determine the single molecule kinetics of wild-type bacteriorhodopsin [2].

To break current resolution limitations, we developed Localization AFM (LAFM). By applying localization image reconstruction algorithms to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius and reach quasi-atomic resolution on soft protein surfaces in native and dynamic conditions. The LAFM method allows the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, opening new avenues for single molecule structural analysis [3].

I will review these recent achievements, and present novel data and methods to integrate and strengthen HS-AFM as a powerful tool in structural biology and molecular biophysics. We reason that the progress of AFM must comprise two axes: First, making AFM/HS-AFM a better tool. With the above-mentioned methods, we made significant progress in this axis over the last ~5 years. Second, making AFM/HS-AFM data an integrated part of the structural biology / molecular biophysics toolbox. Such, we are developing now methods and data analysis and interpretation methods that interface with cryo-EM and X-ray crystallography, where AFM/HS-AFM can contribute the urgently needed information about structure, dynamics and conformations under close-to-native conditions.

References:
[1] Heath et al. Nature Communications, 2018, 9(1):4983, High-Speed AFM Height Spectroscopy (HS-AFM-HS): Microsecond dynamics of unlabeled biomolecules.
[2] Perrino et al., Nature Communications, 2021 12(7225), doi.org/10.1038/s41467-021-27580-2, Single molecule kinetics of bacteriorhodopsin by HS-AFM.
[3] Heath et al. Nature, 2021, 594(7863):385–390, doi:10.1038/s41586-021-03551-x, Localization Atomic Force Microscopy.