Multifrequency atomic force microscopy is used to gain sub-nanometre insights into the structure of aquaporin 0 and its interface with the surrounding water.
Aquaporins are integral membrane proteins that regulate the transport of water and small molecules in and out of the cell. In eye lens tissue, circulation of water, ions and metabolites is ensured by a microcirculation system in which aquaporin-0 (AQP0) plays a central role. AQP0 allows water to flow beyond the diffusion limit through lens membranes. AQP0 naturally arranges in a square lattice. The malfunction of AQP0 is related to numerous diseases such as cataracts. Despite considerable research into its structure, function and dynamics, the interface between the protein and the surrounding liquid and the effect of the lattice arrangement on the behaviour of water at the interface with the membrane are still not fully understood. Here we use a multifrequency atomic force microscopy (AFM) approach to map both the liquid at the interface with AQP0 and the protein itself with sub-nanometer resolution. Imaging using the fundamental eigenmode of the AFM cantilever probes mainly the interfacial water at the surface of the membrane. The results highlight a well-defined region that surrounds AQP0 tetramers and where water exhibits a higher affinity for the protein. Imaging in the second eigenmode is dominated by the mechanical response of the protein and provides sub-molecular details of the protein surface and the sub-surface structure. The relationship between modes and harmonics is also examined.