Dynamic interplay between lateral diffusion and conformational states in a secondary transporter revealed by...

Understanding how membrane proteins coordinate their structural dynamics with their local lipid environment is a crucial process for their function and potential nanotechnological applications. Here, we apply high-speed atomic force microscopy (HS-AFM) to directly visualize, in real time, the lateral motion and substrate-induced conformational changes of the lactose permease (LacY) reconstituted into proteolipid sheets. We observe that LacY dimers diffuse laterally with an average velocity of ∼3 nm s^-1 and a diffusion coefficient of ∼18 nm² s^-1, suggesting that proteins can relocate tens of nanometers before each transport cycle. The addition of lactose and ionophores (valinomycin, nigericin) triggers distinct nanomechanical and morphological responses, revealing a tight coupling between electrochemical gradients, membrane mechanics, and the conformational state of LacY. These findings support the view that transport efficiency is not solely governed by static structure but is dynamically modulated by the protein's lateral mobility and mechanical feedback from the membrane. Our results offer a mechanistic framework for studying secondary transporters in native-like environments and highlight the potential of HS-AFM to resolve nanoscale dynamics in complex membrane systems.