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 transpor ter from Escherichia coli, enzyme IImtl, have been studied by differen tial scanning calorimetry. To this end, the wild type enzyme, IICBA(mt l), as well as IICBmtl and IICmtl, were reconstituted into a dimyristo ylphosphatidylcholine lipid bilayer, The changes in the gel to liquid crystalline transition of the lipid indicated that the protein was ins erted into the membrane, disturbing a total of approximately 40 lipid molecules/protein molecule, The thermal unfolding profile of EIImtl ex hibited three separate transitions, two of which were overlapping, tha t could be assigned to structural domains in the protein. Treatment wi th trypsin, resulting in the degradation of the water-soluble part of the enzyme while leaving the binding and translocation capability of t he enzyme intact, resulted in a decrease of the T-m and enthalpy of un folding of the membrane-embedded C domain. This effect was much more a pparent in the presence of the substrate but only partly so in the pre sence 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 subst rate binding process.