High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties.

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.

Investigating the Mechanisms of AquaporinZ Reconstitution through Polymeric Vesicle Composition for a Biomimetic Membrane.

Aquaporin-Z (AqpZ) are water channel proteins with excellent water permeability and solute rejection properties. AqpZ can be reconstituted into vesicles utilizing cell-like bilayer membranes assembled from amphiphilic block copolymers, for the preparation of high-performance biomimetic membranes. However, only a few copolymers have been found suitable to act as the membrane matrix for protein reconstitution. Hence, this work analyzes the mechanism of protein reconstitution based on a composition-reconstitution relationship. The vesicle formation and AqpZ reconstitution processes in various amphiphilic block copolymers were investigated in terms of size, morphology, stability, polymeric bilayer membrane rigidity, and thermal behavior. Overall, this study contributes to the understanding of the composition-reconstitution relationship of biomimetic membranes based on AqpZ-reconstituted polymeric vesicles.