A significantly higher relative Selleckchem Olaparib fluorescence was detected between SgrT-NGfp and EIICBGlc-CGfp (lane 8) and EIICGlc-linker-CGfp (lane 12), respectively. These results indicate an interaction between SgrT and the full-length protein EIICBGlc or the Linker-EIICGlc-domain, respectively. Significance levels: *p = 0.05, ***p = 0.001. 2.3. The KTPGRED

Motif in the Linker Region of EIICBGlc is the Main SgrT Target Sequence In a previously published experiment, we identified the single amino acid substitution P384R in EIICBGlc, which caused a complete release of SgrT inhibition during growth in minimal medium with glucose as a sole carbon source [27]. The amino Inhibitors,research,lifescience,medical acid P384 is located within the conserved KTPGRED motif. The function of this region was unknown until now, but it seems to play an important role in SgrT regulation. Accordingly, as indicated in Figure 2, lane 13, no bimolecular fluorescence complementation was detected for SgrT-NGfp and

EIICGlc-linker-P384R-CGfp. Inhibitors,research,lifescience,medical To identify other functionally important amino acid residues, we performed SgrT-EIICBGlc crosslinking assays with single amino acid substitutions in the KTPGRED motif of the glucose transporter. All amino acid residues of this motif were replaced by the small Inhibitors,research,lifescience,medical and hydrophobic amino acid residue alanine. In addition, EIICBGlc P384R was also Inhibitors,research,lifescience,medical reanalyzed in this test. The obtained EIICBGlc derivatives were capable

of complementing a ptsG deletion strain on a MacConkey glucose (McCGlc) plate, even EIICBGlc G385A (data not shown). This indicates that under high glucose concentrations (1% in McC plates) the residual activity of all mutants is sufficient to complement transport activity and that all proteins are folded correctly. Similarly important is the fact that all proteins were stable and could be purified easily. Cells overexpressing SgrT Inhibitors,research,lifescience,medical and the respective EIICBGlc derivative were grown in rich medium in the presence of glucose and treated with paraformaldehyde. Subsequently, cells were disrupted found and EIICBGlc-His was purified with Ni-NTA agarose. Respective SgrT co-purifications were visualized by Western blot analysis. As shown in Figure 3A the strongest effect was exhibited by the P384R substitution, which completely abolished the interaction between the two proteins. Strong effects were also caused by the substitutions T383A, P384A, G385A, R386A and E387A. Compared to the wild type protein almost no effects were obtained for the substitutions K382A and D388A. This might indicate that the crucial residues for the EIICBGlc – SgrT interaction are in the center of this sequence motif. Figure 3 Crosslinking experiments with different KTPGRED mutants of EIICBGlc and SgrT.