CLC Antiporter Dimerization Dynamics Revealed by Novel Developments in High-Speed AFM

Abstract

Diffusion of and interactions between membrane proteins in lipid membranes can be vital to their function but quantification of the strength of these interactions has been challenging. Furthermore, these dynamics can occur on a wide range of time- and length-scales rendering them difficult to study. Here we investigate the dimerization of CLC-type transporters through the development and application of high-speed AFM height spectroscopy (HS-AFM-HS). Monitoring the motions of unlabeled proteins underneath the HS-AFM tip at a fixed position with microsecond temporal resolutions allows us to simultaneously measure surface concentrations, diffusion coefficients and oligomeric states of CLC-ec1. These measurements give key kinetics allowing us to describe self-assembly processes in great detail and quantitatively. Combining HS-AFM-HS with HS-AFM high-resolution imaging, we can assess the dynamics and structure of unlabeled bio-molecules with spatial and temporal accuracy spanning several orders of magnitude.