Interdomain interactions in the mannitol permease of E-coli

W Meijberg*, GK Schuurman-Wolters, GT Robillard

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingConference contributionAcademicpeer-review

Abstract

The mannitol permease of E. coli, Enzyme IImannitol, catalyses the concomitant phosphorylation and transport of mannitol across the cytoplasmic membrane. The protein consists of three domains, one N-terminal transmembrane domain (C) and two cytoplasmic domains, A and B. During the catalytic cycle the A and B domains are transiently phosphorylated before the phosphoryl group is transferred to the mannitol. Our research focuses on the characterization of domain interactions using calorimetry and other biophysical techniques. Careful analysis of DSC and GuHCl-induced unfolding data of the binary combination IIBA(mtl) indicated that the B domain is less stable when covalently attached to the A domain than when free in solution and that phosphorylation has a destabilizing effect on the A domain but not on the B domain. It was concluded that strong cooperative interactions are absent between these two domains. This behaviour could be due to several reasons: (i) the need for high phosphorylation rates requires the covalent attachment of the domains; (ii) high flexibility is needed to make the transfer of the phosphoryl group possible; or (iii) the destabilizing interaction is compensated by the interactions between the B and C domains. DSC and spectroscopic methods have been further used to characterize the latter interaction. Two model systems are used, in which the protein is either solubilized in detergent or reconstituted in phospholipid vesicles. In the detergent-solubilized system two overlapping transitions at 59 and 69 degrees C could be observed. The reconstituted system shows comparable behaviour, but in this case both transitions are well separated at 62 and 82 degrees C. From comparison with reconstituted C domain it is clear that the high temperature transition belongs to the C domain. At this moment we are in the process of further assigning the observed transitions to structural entities in the protein in order to determine quantitatively the interactions between the B and C domains in EIImtl.

Original languageEnglish
Title of host publicationBiocalorimetry: Applications of Calorimetry in the Biological Sciences
EditorsE. Ladbury, B.Z. Chowdhry
Place of PublicationCHICHESTER
PublisherWiley
Pages303-314
Number of pages12
ISBN (Print)0-471-97781-0
Publication statusPublished - 1998
EventConference on the Applications of Calorimetry in the Biological Sciences -
Duration: 1-Sep-1996 → …

Other

OtherConference on the Applications of Calorimetry in the Biological Sciences
Period01/09/1996 → …

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