Lipid-Dependent Titration of Glutamic Acid at a Membrane Interface

Abstract

The ionization of the glutamic acid side chain has been predicted to depend on its depth of burial in a lipid-bilayer membrane (Biophys J 94, 3393) but has been difficult to verify experimentally (BBA 1859, 484). To address the issue, we incorporated Glu at position 4 in the transmembrane helix of GWALP23 to yield the peptide acetyl-GGAE4W(LA)6LWLAGA-amide. The parent helix exhibits limited dynamic averaging in bilayer membranes and is tilted from the bilayer normal at a well-defined angle that varies systematically with the membrane thickness (J Biol Chem 285, 31723). Residues A3 and A21 are unwound from the core helix. To detect the titration of residue E4, we labeled the side chain of alanine A3 with deuterium and recorded the 2H NMR spectra for oriented samples of the helix in several lipid-bilayer membranes as a function of pH. Remarkably, the 2H quadrupolar splitting for the side chain of A3 responds to pH with an apparent pKa of 4.8 in DLPC and 6.3 in DMPC but is unchanged up to pH 8.0 in 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 pKa 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.