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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

The pH-dependent chemical shifts of drug-free WT EmrE at 25°C fit to two pKa values, similar to those at 45°C. Proton (A) and nitrogen (B) chemical shifts were plotted independently as a function of pH for both backbone amides and tryptophan side chains (sc). Each y axis is labeled with the residues it represents. The residues are indicated by color as displayed in the figure. The data were globally fit to both a single pKa model (dotted lines; pKa = 6.9 ± 0.1; n = 0.8) and a double pKa model (solid lines; pKa = 6.8 ± 0.1 and 8.5 ± 0.2).
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fig9: The pH-dependent chemical shifts of drug-free WT EmrE at 25°C fit to two pKa values, similar to those at 45°C. Proton (A) and nitrogen (B) chemical shifts were plotted independently as a function of pH for both backbone amides and tryptophan side chains (sc). Each y axis is labeled with the residues it represents. The residues are indicated by color as displayed in the figure. The data were globally fit to both a single pKa model (dotted lines; pKa = 6.9 ± 0.1; n = 0.8) and a double pKa model (solid lines; pKa = 6.8 ± 0.1 and 8.5 ± 0.2).

Mentions: We repeated the pH titration of drug-free EmrE at 25°C to confirm our results, as the pH titration of Glu14 is complicated by tight coupling to global structural and dynamics changes. Changing the temperature helps separate the open-in to open-out conformational exchange processes, which are highly temperature dependent, from protonation/deprotonation of Glu14, which is much less temperature dependent (Chen et al., 2007; Nagai et al., 2008; Pace et al., 2009). At 25°C, slower overall tumbling and changes in the pH-dependent conformational exchange rates between open-in and open-out EmrE lead to increased line broadening, particularly near neutral pH (Figs. 3 B, 5 B, and 6 B). In addition, temperature-dependent chemical-shift changes cause a different subset of residues to be well resolved. As a result, Gly17 is the only residue near Glu14 for which pH-dependent chemical shifts can be measured across the pH titration. Because the 45°C data demonstrated that remote residues accurately sense the Glu14 protonation state, we globally fit the pH-dependent chemical-shift changes of all the well-resolved noncharged residues that were not severely line broadened. The only well-resolved residues that were excluded were His110 and Lys22, as these residues contain titratable side chains. Once again, the data are best fit by two pKa values of 6.8 ± 0.1 and 8.5 ± 0.2 (Fig. 9, solid lines). These are very similar to the pKa values at 45°C, confirming that Glu14 protonation is relatively insensitive to temperature, as expected for a glutamate side chain (Nagai et al., 2008; Smirnova et al., 2008).


Asymmetric protonation of EmrE.

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

The pH-dependent chemical shifts of drug-free WT EmrE at 25°C fit to two pKa values, similar to those at 45°C. Proton (A) and nitrogen (B) chemical shifts were plotted independently as a function of pH for both backbone amides and tryptophan side chains (sc). Each y axis is labeled with the residues it represents. The residues are indicated by color as displayed in the figure. The data were globally fit to both a single pKa model (dotted lines; pKa = 6.9 ± 0.1; n = 0.8) and a double pKa model (solid lines; pKa = 6.8 ± 0.1 and 8.5 ± 0.2).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig9: The pH-dependent chemical shifts of drug-free WT EmrE at 25°C fit to two pKa values, similar to those at 45°C. Proton (A) and nitrogen (B) chemical shifts were plotted independently as a function of pH for both backbone amides and tryptophan side chains (sc). Each y axis is labeled with the residues it represents. The residues are indicated by color as displayed in the figure. The data were globally fit to both a single pKa model (dotted lines; pKa = 6.9 ± 0.1; n = 0.8) and a double pKa model (solid lines; pKa = 6.8 ± 0.1 and 8.5 ± 0.2).
Mentions: We repeated the pH titration of drug-free EmrE at 25°C to confirm our results, as the pH titration of Glu14 is complicated by tight coupling to global structural and dynamics changes. Changing the temperature helps separate the open-in to open-out conformational exchange processes, which are highly temperature dependent, from protonation/deprotonation of Glu14, which is much less temperature dependent (Chen et al., 2007; Nagai et al., 2008; Pace et al., 2009). At 25°C, slower overall tumbling and changes in the pH-dependent conformational exchange rates between open-in and open-out EmrE lead to increased line broadening, particularly near neutral pH (Figs. 3 B, 5 B, and 6 B). In addition, temperature-dependent chemical-shift changes cause a different subset of residues to be well resolved. As a result, Gly17 is the only residue near Glu14 for which pH-dependent chemical shifts can be measured across the pH titration. Because the 45°C data demonstrated that remote residues accurately sense the Glu14 protonation state, we globally fit the pH-dependent chemical-shift changes of all the well-resolved noncharged residues that were not severely line broadened. The only well-resolved residues that were excluded were His110 and Lys22, as these residues contain titratable side chains. Once again, the data are best fit by two pKa values of 6.8 ± 0.1 and 8.5 ± 0.2 (Fig. 9, solid lines). These are very similar to the pKa values at 45°C, confirming that Glu14 protonation is relatively insensitive to temperature, as expected for a glutamate side chain (Nagai et al., 2008; Smirnova et al., 2008).

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