| Abstract: | Fourier transform infrared spectroscopy has been used to study the secondary structure of the human erythrocyte glucose transporter after purification and reconstitution in erythrocyte lipids. The spectra indicate that the glucose transporter contains, in addition to the predominant alpha-helical structure, an appreciable amount of beta-structure and random coil conformation. A study of the time dependency of H-2H exchange revealed that more than 80% of the polypeptide backbone is readily accessible to the solvent. This result indicates that a portion of the intramembrane-spanning region of the membrane protein is exposed to the solvent, suggesting the existence of an intraprotein aqueous channel. The residual (10-20%) portion of the protein which exchanges slowly includes some alpha-helical structure, probably situated in a hydrophobic environment inside the membrane. The infrared spectra of transporter preparations were also examined after incubation with substrate and substrate analogues. Compared with the spectra recorded under conditions in which the "inward-facing" form predominates, a small but reproducible shift in the bands assigned to alpha-helical and beta-strand structures is observed after incubation with 4,6-O-ethylidene-D-glucose, which largely fixes the transporter in the "outward-facing" conformation. An increase of temperature, which is known to increase the proportion of transporter in the outward-facing conformation, results in a similar shift in this alpha-helical absorption band. |
