The thermal stability and domain interactions of the mannitol permease of Escherichia coli. A differential scanning calorimetry study.
The thermal stability and domain interactions in the mannitol transporter from Escherichia coli, enzyme IImtl, have been studied by differential scanning calorimetry. To this end, the wild type enzyme, IICBAmtl, as well as IICBmtl and IICmtl, were reconstituted into a dimyristoylphosphatidylcholine lipid bilayer. The changes in the gel to liquid crystalline transition of the lipid indicated that the protein was inserted into the membrane, disturbing a total of approximately 40 lipid molecules/protein molecule. The thermal unfolding profile of EIImtl exhibited three separate transitions, two of which were overlapping, that could be assigned to structural domains in the protein. Treatment with trypsin, resulting in the degradation of the water-soluble part of the enzyme while leaving the binding and translocation capability of the enzyme intact, resulted in a decrease of the Tm and enthalpy of unfolding of the membrane-embedded C domain. This effect was much more apparent in the presence of the substrate but only partly so in the presence of the substrate analog perseitol. These results are consistent with a recently proposed model (Meijberg, W., Schuurman-Wolters, G. K., and Robillard, G. T. (1998) J. Biol. Chem. 273, 7949-7946), in which the B domain takes part in the conformational changes during the substrate binding process.
Groningen Biomolecular Sciences and Biotechnology Institute and the Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
W
W
Meijberg
G K
GK
Schuurman-Wolters
H
H
Boer
R M
RM
Scheek
G T
GT
Robillard
D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D017931:DNA Primers; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008353:Mannitol / Q000737:chemistry; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D013379:Substrate Specificity
D017931:DNA Primers; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli