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.
© 2021 The Authors. Published by American Chemical Society.