Asymmetric protonation of EmrE

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with (1)H-(15)N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. 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

The mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37.

Solid-state NMR and differential scanning calorimetry were used to investigate the mechanism of lipid bilayer disruption induced by LL-37, an amphipathic alpha-helical, antimicrobial peptide found in humans. The secondary structure, orientation, and dynamics of LL-37 in lipid bilayers were determined using solid-state NMR of site-specifically 13C, 15N, or 2H labeled LL-37. The effect of LL-37 on the lipid molecules was studied with 31P NMR of the lipid headgroup, 2H NMR of the acyl chains, and DSC. The results show that the amphipathic helical region of LL-37 is parallel to the bilayer surface and embedded in the hydrophobic/hydrophilic interface, interacting with both the lipid headgroups and acyl chains. The N-terminal nonhelical region is also associated with the membrane. LL-37 induces positive curvature strain in lipid bilayers, and alters their material properties, such as hydrophobic thickness and area per lipid. In DMPC, LL-37 increases the magnitude of the coefficient of thermal expansion both parallel and perpendicular to the bilayer normal. The orientation of LL-37 is the same regardless of the membrane environment, but the type and extent of headgroup and acyl chain perturbation depends on the lipid charge, headgroup type, acyl chain saturation, presence of cholesterol, and the presence of aqueous ions. This indicates that aggregation of LL-37 into regions of high local concentration and depth of insertion in the membrane interface vary with the relative balance of electrostatic and hydrophobic interactions between the peptide and the lipid headgroups and acyl chains, respectively. Of the mechanisms proposed for bilayer disruption by amphipathic a-helical peptides, the surface orientation and lack of small membrane fragments rule out the barrel-stave and detergent-like micellization mechanisms. All of the results support a toroidal pore model for LL-37 activity in lipid membranes.Ph.D.BiochemistryBiological SciencesBiophysicsPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123977/2/3106185.pd