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Asymmetric protonation of EmrE.

Morrison EA, Robinson AE, Liu Y, Henzler-Wildman KA - J. Gen. Physiol. (2015)

Bottom Line: The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE.Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux.However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus

Gly17 senses the protonation state of EmrE. Enlarged glycine region of the 1H-15N TROSY-HSQC spectrum for WT EmrE. Spectra were collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. Lines highlight the movement of the peaks as the pH is lowered.
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fig6: Gly17 senses the protonation state of EmrE. Enlarged glycine region of the 1H-15N TROSY-HSQC spectrum for WT EmrE. Spectra were collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. Lines highlight the movement of the peaks as the pH is lowered.

Mentions: Closer inspection of the titration patterns of individual residues, particularly Ala10 (Fig. 5) and the glycine region (Fig. 6), reveals that the conformational exchange rate between open-in and open-out states of EmrE is also pH dependent. At high pH, two peaks are observed for each residue of EmrE at 25 and 45°C (Fig. 3). The two peaks present in the spectra of drug-free EmrE at high pH have very similar chemical shifts to the drug-bound spectra of EmrE that we have analyzed previously (Morrison et al., 2012; Morrison and Henzler-Wildman, 2014) and can be assigned to subunit A and subunit B of the asymmetric homodimer. We have shown previously that the two subunits swap conformations as drug-bound EmrE converts from open-in to open-out (Morrison et al., 2012). Whether the two subunits swap conformations at high pH for drug-free EmrE is discussed below in the section, Is apo EmrE still able to interconvert?


Asymmetric protonation of EmrE.

Morrison EA, Robinson AE, Liu Y, Henzler-Wildman KA - J. Gen. Physiol. (2015)

Gly17 senses the protonation state of EmrE. Enlarged glycine region of the 1H-15N TROSY-HSQC spectrum for WT EmrE. Spectra were collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. Lines highlight the movement of the peaks as the pH is lowered.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4664823&req=5

fig6: Gly17 senses the protonation state of EmrE. Enlarged glycine region of the 1H-15N TROSY-HSQC spectrum for WT EmrE. Spectra were collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. Lines highlight the movement of the peaks as the pH is lowered.
Mentions: Closer inspection of the titration patterns of individual residues, particularly Ala10 (Fig. 5) and the glycine region (Fig. 6), reveals that the conformational exchange rate between open-in and open-out states of EmrE is also pH dependent. At high pH, two peaks are observed for each residue of EmrE at 25 and 45°C (Fig. 3). The two peaks present in the spectra of drug-free EmrE at high pH have very similar chemical shifts to the drug-bound spectra of EmrE that we have analyzed previously (Morrison et al., 2012; Morrison and Henzler-Wildman, 2014) and can be assigned to subunit A and subunit B of the asymmetric homodimer. We have shown previously that the two subunits swap conformations as drug-bound EmrE converts from open-in to open-out (Morrison et al., 2012). Whether the two subunits swap conformations at high pH for drug-free EmrE is discussed below in the section, Is apo EmrE still able to interconvert?

Bottom Line: The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE.Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux.However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus