Interdomain interactions between the hydrophilic domains of the mannitol transporter of Escherichia coli in the unphosphorylated and phosphorylated states

Biochemistry
W MeijbergG T Robillard

Abstract

Interdomain interactions in the mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli play a key role in the mechanism of mannitol transport across the membrane [Boer et al. (1995) Biochemistry 34, 3239-3247; Loikema et al. (1991) Biochemistry 30, 6716-6721]. In this study, we focus on the interaction between the hydrophilic A and B domains and try to determine those as a function of the phosphorylation state of the enzyme. To this end, unfolding studies on the subcloned domains IIAmtl and IIBmtl, as well as on the binary combination IIBAmtl, were performed, both in the unphosphorylated and in the phosphorylated states, using GuHCl and heat as the denaturant. It is shown that IIAmtl and IIBmtl, as well as P-IIAmtl and P-IIBmtl, unfold according to a two-state mechanism but that IIBAmtl and P2-IIBAmtl do not exhibit such behavior. Two transitions are observed instead, indicating a lack of strong positive cooperative interactions. DSC studies of the unphosphorylated proteins showed a destabilization of the B domain in IIBAmtl with respect to the free IIBmtl as indicated by a lowereing of the melting temperature and a lower enthalpy of unfolding. Furthermore, it is shown tha...Continue Reading

References

Jul 5, 1974·Journal of Molecular Biology·P L Privalov, N N Khechinashvili
Oct 25, 1981·Journal of Molecular Biology·P L PrivalovL P Revina
Jan 1, 1983·Methods in Enzymology·A Cornish-Bowden
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Sep 1, 1993·Microbiological Reviews·P W PostmaG R Jacobson
Feb 15, 1973·FEBS Letters·N N. KhechinashviliE I. Tiktopulo

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Citations

Nov 25, 1998·Protein Science : a Publication of the Protein Society·C P van MierloE Steensma
Aug 3, 2010·Journal of Surgical Oncology·Martijn F BoomsmaRobert H J Beelen

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