PhD ceremony: Ms. M. Opacic, 16.15 uur, Academiegebouw, Broerstraat 5, Groningen
Title: Structural characterization of the transmembrane domain of the mannitol permease from Escherichia coli using fluorescence and phosphorescence spectroscopy
Promotor(s): prof. B.W. Dijkstra
Faculty: Mathematics and Natural Sciences
In her thesis Milena Opačić presents the structural characterization of the IICmtl domain using fluorescence and phosphorescence spectroscopy. Enzyme IImtl (EIImtl) is a sugar transporter from the inner membrane of Escherichia coli that is responsible for the transport of mannitol from the periplasm to the cytoplasm, and for its phosphorylation. This transporter is part of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). EIImtl is composed ofthree domains: two cytoplasmic domains A and B, and a membrane-embedded domain C (IICmtl). It is functional as a dimer, with one high-affinity mannitol-binding site per dimer.
Förster resonance energy transfer (FRET) study was carried out with nineteen single Trp mutants of EIImtl. The mutants were biosynthetically labelled with 5-fluorotryptophan (5-FTrp), which served as FRET donor. Azi-mannitol, a substrate analogue, was used as acceptor. The study resulted in localization of the mannitol binding site. It was also shown that the binding site is asymmetrically positioned in the EIImtl dimer, and the existence of a second resting binding site was proposed. Furthermore, FRET experiments with stably phosphorylated EIImtl mutants showed that the position of mannitol in IICmtl remains the same as in the unphosphorylated protein. This led to the conclusion that during the mannitol transport cycle, the phosphorylated B domain has to move to the binding site in IICmtl .
5-FTrp fluorescence spectroscopy was also used for structural studies of thirteen single Trp mutants of EIImtl, all of them having the Trp introduced in the IICmtl domain. The emission spectra, the sensitivity for an external quencher, the fluorescence lifetimes and the anisotropy indicated that the 5-FTrps in all mutants are in buried and structured positions. CD spectroscopy showed that IICmtl contains 14 ± 4% β-sheet structure, a feature not observed for other helical membrane proteins. Moreover, when compared with the 2D projections of other membrane transporters, the average area occupied by one transmembrane helix of IICmtl is considerably larger. Taken together, this suggests that the structural organization of IICmtl differs from the structures of membrane transport proteins as yet elucidated.
Phosphorescence spectroscopy, a very sensitive tool to probe the Trp microenvironment, was employed to explore the structure near six positions in IICmtl (38, 180, 251, 260, 282, and 327). Emphasis was placed on positions 251 and 260 flanking the GIXE motif, which is conserved in all EII sugar transporters and whose function is not known. The Trp residues at positions 251 and 260 were shown to be in a polar and apolar microenvironment, respectively. Mannitol binding induced only minor changes in their microenvironment, indicating that mannitol does not bind in close proximity to these residues. However, phosphorylation of the cytoplasmic B domain of EIImtl induced a large structuring at Trp260, but less at Trp251. Because the GIXE motif is predicted to be part of a putative transmembrane helix, our results suggest that this helix is oriented with its C-terminus towards the cytoplasm. Since mannitol binding induced changes in the phosphorescence properties of the positions 180, 282 and 327, which are located >16 Å from the mannitol binding site, long range conformational effects of mannitol binding are proposed.
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