title: Substrate-induced conformational changes in the membrane-embedded IIC(mtl)-domain of the mannitol permease from Escherichia coli, EnzymeII(mtl), probed by tryptophan phosphorescence spectroscopy.
abstract: Membrane-bound transport proteins are expected to proceed via different conformational states during the translocation of a solute across the membrane. Tryptophan phosphorescence spectroscopy is one of the most sensitive methods used for detecting conformational changes in proteins. We employed this technique to study substrate-induced conformational changes in the mannitol permease, EnzymeII(mtl), of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Ten mutants containing a single tryptophan were engineered in the membrane-embedded IIC(mtl)-domain, harboring the mannitol translocation pathway. The mutants were characterized with respect to steady-state and time-resolved phosphorescence, yielding detailed, site-specific information of the Trp microenvironment and protein conformational homogeneity. The study revealed that the Trp environments vary from apolar, unstructured, and flexible sites to buried, highly homogeneous, rigid peptide cores. The most remarkable example of the latter was observed for position 97, because its long sub-second phosphorescence lifetime and highly structured spectra in both glassy and fluid media imply a well defined and rigid core around the probe that is typical of beta-sheet-rich structural motifs. The addition of mannitol had a large impact on most of the Trp positions studied. In the case of position 97, mannitol binding induced partial unfolding of the rigid protein core. On the contrary, for residue positions 126, 133, and 147, both steady-state and time-resolved data showed that mannitol binding induces a more ordered and homogeneous structure around these residues. The observations are discussed in context of the current mechanistic and structural model of EII(mtl).
authors.affiliation: Department of Biochemistry and Biophysical Chemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Gertjan
authors.initials: G
authors.lastname: Veldhuis
authors.forename: Edi
authors.initials: E
authors.lastname: Gabellieri
authors.forename: Erwin P P
authors.initials: EP
authors.lastname: Vos
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
authors.forename: Giovanni B
authors.initials: GB
authors.lastname: Strambini
authors.forename: Jaap
authors.initials: J
authors.lastname: Broos
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers / Q000737:chemistry; D008353:Mannitol / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry*; D010758:Phosphorus / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry / Q000379:methods*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D010758:Phosphorus; D008353:Mannitol; D014364:Tryptophan; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli