Dynamics of membrane proteins using High-Speed Atomic Force Microscopy

Cell membranes are dynamic and complex structures that regulate fundamental biological processes through their interactions with proteins. Both the physical properties of membranes and the conformational flexibility of proteins play central roles in determining function. This thesis applies atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to investigate protein dynamics at high spatial and temporal resolution, with a focus on membrane curvature, peripheral enzymes, and transmembrane ion channels.
First, nanoparticle-supported curved membranes were developed as platforms to mimic nanoscale curvature. Supported lipid bilayers were formed on polystyrene beads, gold nanoparticles, and gold nanorods under varied conditions. AFM imaging and quantitative analysis showed that particle geometry and surface chemistry strongly influence bilayer formation, with gold nanoparticles offering the most stable coverage. These systems provide a controlled means of studying curvature-dependent protein behaviour.
Second, the membrane-modifying enzyme phosphatidylserine decarboxylase (PSD) was examined on supported bilayers. AFM and HS-AFM revealed that PSD preferentially associates with membrane edges and curved regions, displaying dual roles as both an enzyme and a structural organiser. On PScontaining bilayers, active PSD produced ordered ripple-like assemblies, whereas inactive PSD generated disordered spiral-like structures, confirming that enzymatic activity is required for organised remodelling. These findings suggest PSD contributes not only to lipid metabolism but also to the physical regulation of membrane architecture.
Finally, TRPC5, a calcium-permeable ion channel, was studied in reconstituted lipid bilayers. HS-AFM captured tetrameric TRPC5 assemblies
consistent with cryo-EM dimensions, and rare subunit dissociation events suggestive of reversible symmetry changes. Pharmacological modulation by Englerin A and Pico145 showed height changes, while activation induced multiple oligomeric forms, including pentamers, similar to observations inTRPV3 [2] . These results provide new insights into the structural dynamics of TRPC5 under near-native conditions.
Overall, this work demonstrates how AFM-based approaches can bridge static structural data with dynamic membrane behaviour, offering new
perspectives on protein-lipid interaction with implications for biophysics and therapeutic discovery.