Isomerization of photo-Gb(3) in phase-separated pore-spanning membranes alters Shiga toxin organization.

The lateral organization of Shiga toxin bound to a lipid membrane is significantly influenced by the fatty acid geometry of its receptor glycolipid globotriaosylceramide (Gb(3)), which is crucial for the protein's internalization into the host cell. To control the lipid geometry, we used a photoisomerizable azobenzene derivative of Gb(3) (photo-Gb(3)) that can be switched between a trans- and a cis-configuration under light. We reconstituted this photo-Gb(3) into liquid-disordered (l(d))/liquid-ordered (l(o)) phase-separated pore-spanning membranes (PSMs), creating freestanding bilayer parts (freestanding PSMs [f-PSMs]) composed of an l(o) phase surrounded by an l(d) phase on the pore rims. Upon UV irradiation, we observed small, mobile, and transient l(d) domains in the f-PSMs, indicating a trans-to-cis isomerization of the photo-Gb(3). The mean diffusion coefficient of the l(d) domains was determined to be 0.014 ± 0.009 μm(2)/s, from which we estimated a membrane surface viscosity of 12-21 × 10(-8) Pa s m, indicative of an l(o) phase. Before photoisomerization, Shiga toxin B (STxB) bound homogeneously to the l(o) phase in the f-PSMs harboring the photo-Gb(3). Upon trans-to-cis isomerization of the photo-Gb(3), l(d) lakes were again formed, and the homogeneous protein distribution turned into dynamic STxB density fluctuations, which are discussed in the context of the entry process of Shiga toxin into the host cell.