Dynamics of Membrane Proteins Using High-Speed Atomic Force Microscopy

Abstract

Cell membranes are dynamic, heterogeneous platforms whose interactions with proteins are essential for numerous biological processes. Understanding these interactions requires approaches that capture both structural detail and real-time behaviour. In this thesis, atomic force microscopy (AFM) and high-speed AFM (HS-AFM) were used to investigate how membrane curvature, peripheral enzymes, and transmembrane ion channels shape membrane organization and function. First, nanoparticle-supported lipid bilayers were developed to model nanoscale membrane curvature. By forming bilayers on polystyrene beads, gold nanoparticles, and gold nanorods, this work demonstrates that particle geometry and surface chemistry strongly influence membrane coverage, with gold nanoparticles providing the most stable platforms. These curvature-defined systems establish a controlled environment for studying curvature-dependent protein dynamics. Finally, the calcium-permeable ion channel TRPC5 was studied in reconstituted lipid bilayers. HS-AFM resolved tetrameric TRPC5 consistent with cryo-EM structures and captured infrequent subunit dissociation events, suggesting reversible symmetry changes. Pharmacological modulators, including Englerin A and Pico145, produced measurable height changes, while activation led to multiple oligomeric states, including pentamers similar to those observed in TRPV3. Together, these studies show how AFM-based imaging can connect static structural information with dynamic membrane behaviour. By integrating analyses of curvature, enzymatic remodeling, and channel flexibility, this work provides new insight into protein–lipid interactions with implications for membrane biophysics and therapeutic discovery.