Buried lysine, but not arginine, titrates and alters transmembrane helix tilt

Abstract

The ionization states of individual amino acid residues of membrane proteins are difficult to decipher or assign directly in the lipid-bilayer membrane environment. We address this issue for lysines and arginines in designed transmembrane helices. For lysines (but not arginines) at two locations within dioleoyl-phosphatidylcholine bilayer membranes, we measure pK(a) values below 7.0. We find that buried charged lysine, in fashion similar to arginine, will modulate helix orientation to maximize its own access to the aqueous interface or, if occluded by aromatic rings, may cause a transmembrane helix to exit the lipid bilayer. Interestingly, the influence of neutral lysine (vis-à-vis leucine) upon helix orientation also depends upon its aqueous access. Our results suggest that changes in the ionization states of particular residues will regulate membrane protein function and furthermore illustrate the subtle complexity of ionization behavior with respect to the detailed lipid and protein environment.

Fig. 1.

Models to show peptide helix transmembrane orientations in DOPC bilayers as function of lysine residue position and ionization state. (A) Y⁵GWALP23-K⁺14. (B) Y⁵GWALP23-K⁰14. (C) Y⁵GWALP23-K⁰12. (D) Y⁵GWALP23 itself, with no ionizable residue. See text for the peptide sequences. The side chain of residue 12 is shown as sticks for either leucine (A, B, and D) or lysine (C). [Note that Y⁵GWALP23-K⁺12 exhibits multistate behavior (as does GWALP23-R⁺12 (17)) and would not have a single orientation.] The peptide GWALP23-R⁺14 (17) orients similarly to Y⁵GWALP23-K⁺14, shown in (A). Note that Y⁵GWALP23-K⁰12 (C) orients similarly to Y⁵GWALP23 itself (D). An alternate perspective (top view) is shown in (A2–D2). See also the precession of tilted peptides about the membrane normal, shown in Movie S1.

Fig. 2.

²H NMR spectra for labeled alanines 15 (50–70% deuterated) and 17 (100% deuterated) in peptides incorporated into hydrated, oriented bilayers of DOPC. (A) Y⁵GWALP23-K14 at pH 5.2. (B) Y⁵GWALP23-K14 at pH 8.2. (C) Y⁵GWALP23-K12 at pH 5.2 (showing multistate behavior). (D) Y⁵GWALP23-K12 at pH 8.2.

Fig. 3.

Quadrupolar wave analysis of tilted transmembrane peptides. (A) Y⁵GWALP23-K14 at pH 5.2 (red; tilt τ = 15°, rotation ρ = 228°) and pH 8.2 (blue; tilt τ = 9°, rotation ρ = 244°). (B) Y⁵GWALP23-K12 at pH 8.2 (blue; tilt τ = 5°, rotation ρ = 308°). The black curves in (A) and (B) represent Y⁵GWALP23 (tilt τ = 5°, rotation ρ = 311°, irrespective of pH). (C) rmsd plots for tilt and rotation of Y⁵GWALP23 (black), Y⁵GWALP23-R14 (orange), Y⁵GWALP23-K14 (red, pH 5.2), and Y⁵GWALP23-K14 (blue, pH 8.2). (D) rmsd plot for tilt and rotation of Y⁵GWALP23-K12 at pH 8.2. Contours are drawn at 1, 2, and 3 kHz.

Fig. 4.

(A) Titration curve for Y⁵GWALP23-K14 in oriented DOPC bilayer membranes at 50 °C, based on the ²H quadrupolar splittings for deuterated A15 (black squares) and A17 (red circles). Both datasets agree on a pK_a of 6.2 at 50 °C (dashed vertical segment). When residue 14 is arginine (dashed red segment; A17 data) or leucine (dashed blue segment), the titration behavior is not observed. (B) pH dependence of the main peak intensities for the ²H NMR resonances of labeled A15 and A17 of Y⁵GWALP23-K12 in DOPC. A 50% maximal peak intensity is achieved for both alanines at pH 7.0; 50 °C. The solid-state ²H NMR spectra are included in Figs. S2 and S3.