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.
Highlights
Fourier transform infrared spectroscopy has been reversible inhibitor of glucose transport, with the same high used to study the secondary structure of the human affinity and sensitivity to competition by D-glucose as in the erythrocyte glucose transporter after purification and cell [7]
Compared with the spectra recorded under conditions 4,6-O-ethylidene-~-glucoasen,ontransported glucose derivain which the “inward-facing” form predominates, a tive,hasa 10-fold higher affinity for the outward facing small but reproducible shift inthe bands assigned to0- conformation than for the inward-facing conformation of the helical and @-strand structuriessobserved after incu- transporter [17, 18]
We investigate the magnitude of the conformational changes associated with glucose transport
Summary
Outdated human bloodwas provided by the blood bank of the Royal Free Hospital. Endoglycosidase F (endo-8-N-acetyl glucosaminidase F) was a generous gift of Boehringer Mannheim. The FT-IRspectrum of the glucose transporter after purification andreconstitution in erythrocyte lipids in H 2 0 is shown in Fig. 1.The difference spectrum reveals a phospholipid carbonyl stretching absorption at 1734 cm" and amide I and amide I1 bands at 1656 and 1547cm", respectively (Fig. la). Transporter samples were nents at 1544, 1528, and 1516 cm" These absorption bands prepared in 'HZO either by dialyzing the glucose transporter against can be demonstrated by deconvolution of the difference 'The abbreviations and trivial name used are: FT-IR, Fourier transform infrared; SDS, sodium dodecyl sulfate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid endoglycosidase F, endo-B-N-acetyl glucosaminidase; ethylidene-D-glucose,4,6-0-ethyspectrum, as shown in Fig. Id, the bands are not so well defined as in the second- and fourth-derivative spectra. Lidene-a-D-glucose;propyl glucoside, n-propyl-8-D-glucopyranoside. 2a) shows the carbonyl stretching vibration at 1733 cm" and FSTt-uIdRy of HuEmryatnhrocyte
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