Maltoporins are a family of membrane proteins that facilitate the diffusion of hydrophilic molecules and maltosaccharides across the outer membrane of Gram-negative bacteria. Two contradicting models propose the sugar binding, uptake, and transport by maltoporins to be either symmetric or asymmetric. Here, we address this contradiction and introduce force-distance-based atomic force microscopy to image single maltoporin LamB trimers in the membrane at sub-nanometer resolution and simultaneously quantify the binding of different malto-oligosaccharides. We assay subtle differences of the binding free-energy landscape of maltotriose, maltotetraose, and maltopentaose, which quantifies how binding strength and affinity increase with the malto-oligosaccharide chain length. The ligand-binding parameters change considerably by mutating the extracellular loop 3, which folds into and constricts the transmembrane pore of LamB. By recording LamB topographs and structurally mapping binding events at sub-nanometer resolution, we observe LamB to preferentially bind maltodextrin from the periplasmic side, which shows sugar binding and uptake to be asymmetric. The study introduces atomic force microscopy as an analytical nanoscopic tool that can differentiate among the factors modulating and models describing the binding and uptake of substrates by membrane proteins.
Keywords: Functional imaging; high-resolution atomic force microscopy; ligand-binding free-energy landscape; maltoporin; outer membrane protein; sugar transport and binding.