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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.

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

No MeSH data available.


Related in: MedlinePlus

Drug-free EmrE is sensitive to pH. Full 1H-15N TROSY-HSQC spectra of drug-free WT EmrE in DLPC/DHPC isotropic bicelles at three pH values collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. All spectra in the full pH titration datasets are shown in Figs. S1 (45°C) and S3 (25°C). Circles highlight several residues that remain in the slow exchange regime at 25°C at low pH.
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fig3: Drug-free EmrE is sensitive to pH. Full 1H-15N TROSY-HSQC spectra of drug-free WT EmrE in DLPC/DHPC isotropic bicelles at three pH values collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. All spectra in the full pH titration datasets are shown in Figs. S1 (45°C) and S3 (25°C). Circles highlight several residues that remain in the slow exchange regime at 25°C at low pH.

Mentions: Ideally, the pKa of ionizable residues is measured by following the 13C and 1H chemical shifts of the side-chain carbons that are closest to the ionizable group in each titratable residue (Lindman et al., 2006; Platzer et al., 2014). Unfortunately, we were not able to do this because of technical challenges. Limited side-chain assignments and chemical-shift overlap in 1H-13C HSQC spectra prevent direct monitoring of the Glu14 side chain in uniformly labeled EmrE. Amino acid–specific labeling of glutamate when EmrE is expressed in its native E. coli is precluded as a result of the central role of glutamate in metabolism. Elegant NMR experiments designed to selectively monitor aspartate and glutamate side chains (Pellecchia et al., 1997; Castañeda et al., 2009) in uniformly labeled samples did not have sufficient sensitivity when applied to EmrE solubilized in isotropic bicelles. Therefore, we have used simple 1H-15N TROSY-HSQC spectra to monitor the pH-dependent chemical shift of backbone amides in EmrE (Figs. 3 and S1).


Asymmetric protonation of EmrE.

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

Drug-free EmrE is sensitive to pH. Full 1H-15N TROSY-HSQC spectra of drug-free WT EmrE in DLPC/DHPC isotropic bicelles at three pH values collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. All spectra in the full pH titration datasets are shown in Figs. S1 (45°C) and S3 (25°C). Circles highlight several residues that remain in the slow exchange regime at 25°C at low pH.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Drug-free EmrE is sensitive to pH. Full 1H-15N TROSY-HSQC spectra of drug-free WT EmrE in DLPC/DHPC isotropic bicelles at three pH values collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. All spectra in the full pH titration datasets are shown in Figs. S1 (45°C) and S3 (25°C). Circles highlight several residues that remain in the slow exchange regime at 25°C at low pH.
Mentions: Ideally, the pKa of ionizable residues is measured by following the 13C and 1H chemical shifts of the side-chain carbons that are closest to the ionizable group in each titratable residue (Lindman et al., 2006; Platzer et al., 2014). Unfortunately, we were not able to do this because of technical challenges. Limited side-chain assignments and chemical-shift overlap in 1H-13C HSQC spectra prevent direct monitoring of the Glu14 side chain in uniformly labeled EmrE. Amino acid–specific labeling of glutamate when EmrE is expressed in its native E. coli is precluded as a result of the central role of glutamate in metabolism. Elegant NMR experiments designed to selectively monitor aspartate and glutamate side chains (Pellecchia et al., 1997; Castañeda et al., 2009) in uniformly labeled samples did not have sufficient sensitivity when applied to EmrE solubilized in isotropic bicelles. Therefore, we have used simple 1H-15N TROSY-HSQC spectra to monitor the pH-dependent chemical shift of backbone amides in EmrE (Figs. 3 and S1).

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