HS-AFM Applications to Study Membrane Proteins.

High-speed atomic force microscopy (HS-AFM) has emerged as a powerful tool for the investigation of the dynamic behavior of membrane proteins in near-native environments. Unlike traditional structural techniques, HS-AFM allows for real-time imaging of individual protein molecules at nanometer resolution under physiological conditions. This capability enables direct visualization of conformational changes, intermolecular interactions, and functional cycles of membrane proteins, such as channels, transporters, and receptors, without the need for labeling or extensive sample preparation. HS-AFM provides insights into the structural plasticity and mechanistic pathways of membrane proteins that are often averaged out or missed by ensemble techniques. Its ability to capture dynamic events at sub-second timescales has significantly advanced our understanding of membrane protein conformational dynamics, single-molecule kinetics, as well as about diffusion, clustering, and interactions within lipid bilayers. As the technology continues to evolve, HS-AFM holds great promise for bridging structural and functional studies, offering a unique window into the real-time molecular mechanisms of membrane-associated biological processes. In this chapter, we detail protocols from membrane protein expression, purification, and reconstitution to final HS-AFM experiments.