Part II: Diffraction from Two-Dimensional Cholera Toxin Crystals Bound to Their Receptors in a Lipid Monolayer

The structure of cholera toxin (CTAB5) bound to its putative ganglioside receptor, galactosyl-N-acetylgalactosaminyl (N-acetyl-neuraminyl) galactosylglucosylceramide (GM1), in a lipid monolayer at the air-water interface has been studied utilizing grazing incidence x-ray diffraction. Cholera toxin is one of very few proteins to be crystallized in two dimensions and characterized in a fully hydrated state. The observed grazing incidence x-ray diffraction Bragg peaks indicated cholera toxin was ordered in a hexagonal lattice and the order extended 600–800 Å. The pentameric binding portion of cholera toxin (CTB5) improved in-plane ordering over the full toxin (CTAB5) especially at low pH. Disulfide bond reduction (activation of the full toxin) also increased the protein layer ordering. These findings are consistent with A-subunit flexibility and motion, which cause packing inefficiencies and greater disorder of the protein layer. Corroborative out-of-plane diffraction (Bragg rod) analysis indicated that the scattering units in the cholera layer with CTAB5 shortened after disulfide bond reduction of the A subunit. These studies, together with Part I results, revealed key changes in the structure of the cholera toxin-lipid system under different pH conditions.