Lipid-Dependent Titration of Glutamic Acid at a Bilayer Membrane Interface

Abstract

The ionization properties of protein side chains in lipid-bilayer membranes will differ from the canonical values of side chains exposed to an aqueous solution. While the propensities of positively charged side chains of His, Lys, and Arg to release a proton in lipid membranes have been rather well characterized, the propensity for a negatively charged Glu side chain to receive a proton and achieve the neutral state in a bilayer membrane has been less well characterized. Indeed, the ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid membrane but has been difficult to verify experimentally. To address the issue, we incorporated an interfacial Glu residue at position 4 of a distinct 23-residue transmembrane helix and used ²H NMR to examine the helix properties as a function of pH. We observe that the helix tilt and azimuthal rotation vary little with pH, but the extent of helix unraveling near residues 3 and 4 changes as the Glu residue E4 titrates. Remarkably, the ²H quadrupolar splitting for the side chain of alanine A3 responds to pH with an apparent pK _a of 4.8 in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 6.3 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), but is unchanged up to pH 8.0 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the presence of residue E4. With bilayers composed of alkali-stable ether-linked lipids, the side chain of A3 responds to pH with an apparent pK _a of 11.0 in the ether analogue of DOPC. These results suggest that the depth dependence of Glu ionization in lipid-bilayer membranes may be steeper than previously predicted or envisioned.

Figure 1

Tilted model of the helix of E⁴GW^5,19ALP23 in a bilayer membrane, highlighting the side chains of residues A3, E4, W5, and W19.

Figure 2

²H NMR spectra for labeled alanine A3 of E⁴GW^5,19ALP23 in mechanically aligned bilayers of DLPC, DMPC, and DOPC, oriented at β = 90 and 0°, temperature 50 °C, and sample pH as indicated. For experiments above pH 8, the membrane was made from the ether-linked analogue of DOPC.

Figure 3

(A) ²H NMR spectra for deuterated core alanines A7 (50% ²H) and A9 (100% ²H) in E⁴GW^5,19ALP23 in DMPC or DOPC lipid bilayers at pH 3 and 8. (B) ²H NMR spectra for deuterated alanine A5 of E⁴A⁵GW¹⁹ALP23 in DLPC bilayers at pH 3 and 8. Each sample is oriented at β = 90°, and the temperature is 50 °C.

Figure 4

Titration curves monitoring the side-chain CD₃ quadrupolar splitting of alanine A3 in E⁴GW^5,19ALP23 within oriented bilayers of DLPC (black), DMPC (blue), and DOPC (red). The midpoints of the curves indicate pK_a values of 4.8 (DLPC), 6.3 (DMPC), and 11.0 (DOPC).

Figure 5

GALA wave plots to compare the core helix orientations for GWALP23 when E4 or L4 is present. Quadrupolar wave plots are presented for the helices in DLPC, DMPC, and DOPC, as indicated. In each membrane, the core helix tilt changes when L4 (black curves) is changed to E4 but does respond to the ionization of E4 (blue and red data points). When E4 titrates in DLPC, the extent of the unraveling of residue 3 changes, and residue 7 also becomes unwound from the core helix. When E4 titrates in DMPC or DOPC, residue 3 again responds, but residue 7 remains on the curve for the core helix. In DOPC, residue 3 is on the curve for the parent core helix with L4 but unravels when E4 is introduced and deviates still further when E4 titrates.