Abstract
Secondary transporters exist as monomers, dimers or higher state oligomers. The significance of the oligomeric state is only partially understood. Here, the significance of the trimeric state of the L-carnitine/γ-butyrobetaine antiporter CaiT of Escherichia coli was investigated. Amino acids important for trimer stability were identified and experimentally verified. Among others, CaiT-D288A and -D288R proved to be mostly monomeric in detergent solution and after reconstitution into proteoliposomes, as shown by blue native gel electrophoresis, gel filtration, and determination of intermolecular distances. CaiT-D288A was fully functional with kinetic parameters similar to the trimeric wild-type. Significant differences in amount and stability in the cell membrane between monomeric and trimeric CaiT were not observed. Contrary to trimeric CaiT, addition of substrate had no or only a minor effect on the tryptophan fluorescence of monomeric CaiT. The results suggest that physical contacts between protomers are important for the substrate-induced changes in protein fluorescence and the underlying conformational alterations.
Highlights
CaiT is a L-carnitine/γ-butyrobetaine antiporter found in the cytoplasmic membrane of E. coli and other Enterobacteriaceae[1]
While the long C-terminal domain of BetP is proposed to participate in inter-protomer interaction[16,17], the respective domain of CaiT is with only two amino acids too short to interact with another protomer
Amino acids of the long, curved α-helix 7 and the periplasmic end of transmembrane domains (TMDs) 2 seem to participate in inter-protomer interactions (Table S1 and Fig. S1)
Summary
CaiT is a L-carnitine/γ-butyrobetaine antiporter found in the cytoplasmic membrane of E. coli and other Enterobacteriaceae[1]. BetP is involved in adaptation of bacteria to osmotic stress and functions as transporter and as a regulator of its own activity[11]. The crystal structures of CaiT do not reveal significant conformational differences between the protomers in the trimer[13,14], and the significance of trimer formation by CaiT is not known. BetP loses its function as a regulator when in an engineered monomeric state probably due to altered protein interactions of the extended N- and/or C-terminal domains performing this regulation[18]. We set out to obtain information on the significance of the oligomeric state of CaiT that despite its structural similarity to BetP is Na+-independent and lacks the N- and C-terminal domains involved in regulation. Differences in the effect of substrate binding on tryptophan fluorescence between monomeric and trimeric CaiT suggest that a physical contact between the protomers of a trimer has an influence on conformational alterations associated with substrate binding
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
