Abstract
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.
Highlights
The thermal stability and domain interactions in the mannitol transporter from Escherichia coli, enzyme IImtl, have been studied by differential scanning calorimetry
The effect is even stronger in the unfolded state, since unfolding results in exposure to the solvent of hydrophobic parts that are normally buried in the interior of the protein. We circumvented this problem by reconstituting EIImtl, as well as IICBmtl and IICmtl, in a lipid bilayer, enabling us to study the thermal stability of the enzyme by differential scanning calorimetry and CD spectroscopy
We focus on the B and C domains in order to get better insight into their mutual interactions
Summary
(Received for publication, December 23, 1997, and in revised form, June 4, 1998). Wim Meijberg, Gea K. 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. EIImtl is well suited for this type of study, since the individual domains of EIImtl are available in subcloned form and are enzymatically active and, are expected to retain their structural integrity It is obvious from what is known about the catalytic mechanism of EIImtl that domain interactions play an important role in the functioning of the enzyme. We focus on the B and C domains in order to get better insight into their mutual interactions
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