Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity

Abstract

High amphiphilicity is a hallmark of interfacial helices in membrane proteins and membrane-active peptides, such as toxins and antimicrobial peptides. Although there is general agreement that amphiphilicity is important for membrane-interface binding, an unanswered question is its importance relative to simple hydrophobicity-driven partitioning. We have examined this fundamental question using measurements of the interfacial partitioning of a family of 17-residue amidated-acetylated peptides into both neutral and anionic lipid vesicles. Composed only of Ala, Leu, and Gln residues, the amino acid sequences of the peptides were varied to change peptide amphiphilicity without changing total hydrophobicity. We found that peptide helicity in water and interface increased linearly with hydrophobic moment, as did the favorable peptide partitioning free energy. This observation provides simple tools for designing amphipathic helical peptides. Finally, our results show that helical amphiphilicity is far more important for interfacial binding than simple hydrophobicity.

Figure 1

Amphipathic helices at membrane interfaces. Residues are colored according to residue type: yellow, non-polar; green, polar; blue, basic; red, acidic. The non-polar residues generally face the hydrocarbon interior of the bilayer while the polar and charged residues face the aqueous phase. (a) Melittin, an archetypal toxin peptide, embedded in the interface of a dioleoylphosphatidylcholine (DOPC) bilayer. The image was created from a frame taken from a restrained molecular dynamics simulation. (b) Channel domain of the KirBac1.1 ligand-gated K⁺-channel in the closed state, including the interfacial slide helices that mechanically couple the ligand receptor (not shown) to channel opening. Amphipathic helices such as these are common structural features of membrane proteins,. Images produced with VMD software.

Figure 2

Thermodynamic schemes for peptide partitioning and folding in membrane interfaces. (a) Four-state thermodynamic cycle for describing the partitioning and folding of peptides into bilayer interfaces from water using four states: fully unfolded peptide in the aqueous phase (state A) and in the membrane (state B), partially folded in aqueous phase (state C), and folded in the membrane interface (state D). This scheme takes the fully unfolded peptide in the aqueous phase as the reference state. As we state before, measurements of the partitioning of most biologically interesting peptides yield free energies for the C to D equilibrium, because the B state is much less populated than the D state. Nevertheless, the A to B equilibrium establishes an important reference state. (b) Two-state thermodynamic cycle commonly used for describing peptide partitioning into the membrane interface. Three thermodynamic states are shown: partially folded in water (state C), partially folded in membrane (state C’), and folded in the membrane interface (state D). The C’ state cannot generally be observed experimentally by the usual optical methods because its occupancy is very small compared to state D. This thermodynamic scheme is a very practical one, because the C and D states (and therefore ΔG_CD) are readily accessible by circular dichroism and fluorescence measurements. But the difficulty is that each peptide has a different degree of folding in the aqueous and membrane phases, which complicates the per-residue folding free energy in the interface. The scheme in panel a avoids this problem by tying all measures to a common reference state.

Figure 3

CD spectra of the peptides A₈Q₃L₄-5.51, A₈Q₃L₄-2.86, and A₈Q₃L₄-0.55 at 25 °C in 10 mM phosphate buffer, and 20–30 μM peptide. The spectra were taken in a 1-mm path length cuvette and averaged over 10–20 scans. Increasing the hydrophobic moment increases the helical content in water (measured as molar ellipticity at 222 nm).

Figure 4

Helicity of the A₈Q₃L₄-family of peptides in water and the membrane interface (POPC solid down-side triangles and POPC:POPG open down-side triangles) as a function of hydrophobic moment. Helicity is a linear function of hydrophobic moment in both media, and it does not depend on the lipid surface charge. Helicity in the membrane interface was determined from [Θ_max] computed from binding curves. See Methods and Supplementary Figure S3.

Figure 5

The linear dependence of the free energy of helix formation in water as a function of hydrophobic moment.

Figure 6

The free energy of partitioning (ΔG_CD) the A₈Q₃L₄-family of peptides into POPC (solid red circles) and POPC:POPG (solid black squares) LUV interfaces. The solid line is the best-fit linear curve through all points. Partitioning free energy values for TMX-3 (31-residues: GWAALAAHAAPALAAALAHAAASRSRSR-amide; μ_H = 3.32 at pH 7.6 and 4.25 at pH 6) and melittin (26 residues: GIGAVLKVLTTGLPALISWIKRKRQQ-amide; μ_H = 5.18) are included for comparison (closed and open circles, respectively). The TMX-3 and melittin free energies are not described particularly well by the linear curve, which is not surprising given the great differences in sequence and length compared to the A₈Q₃L₄-family. More important, however, is the fact that both TMX-3 and melittin have little tendency to partition into POPC interfaces based upon total hydrophobicity (see text).

Figure 7

Free energies of folding of the A₈Q₃L₄-family of peptides in the POPC interface. (a) Values of free energy ΔG_BD of folding in the POPC interface computed using the thermodynamic scheme of Figure 2a and the computed values for ΔG_AB (Table 1, ΔG_AB ≡ ΔG_WW) and the measured values of ΔG_AC and ΔG_CD. The corresponding values of melittin and TMX-3 (solid circle and solid square, respectively) fall far off the curve. This appears in large measure to be due to sequence length. (b) The data of panel a re-plotted using per-residue free energies ΔG_residue computed from ΔG_residue = ΔG_BD/f_αn, where f_α is the fractional helicity and n is the number of residues in the sequence. Notice that the TMX-3 and melittin data are described reasonably well by the A₈Q₃L₄ when length is accounted for, computed as follows: Melittin has a 6% helix content in aqueous solution that increases to f_α = 0.71 when membrane-bound,. We determined experimentally that ΔG_AC = 1.62(±0.06) kcal mol⁻¹ (data not shown), and computed from the Hristova-White algorithm computed that ΔG_AB = −0.07 kcal mol⁻¹. From these values, the value of ΔG_CD (see text), and n = 26, ΔG_residue is found to be −0.27(±0.01) kcal mol⁻¹ per residue. TMX-3 has a 22% helix content in aqueous solution, which increases to about 70% when membrane–bound at neutral pH. We determined experimentally (data not shown) that ΔG_AC = 0.74(±0.03) kcal mol⁻¹. From these values, the value of ΔG_CD (see text), and n = 31, we computed ΔG_residue as −0.24(±0.01) kcal mol⁻¹ for TMX-3 in the deprotonated form. This value is approximate, because ΔG_AC was determined only at neutral pH where the amino terminus and the His residues are still partially protonated.

Figure 8

The free energy of partitioning of the A₈Q₃L₄ family of peptides from buffer to the POPC bilayer interface plotted as a function of the difference in helicity (Δf_α) of the peptides in the membrane and in water (Figure 4).

Figure 9

The helicity of the A₈Q₃L₄ family of peptides in membranes f_mα plotted against the fractional helicities f_wα in buffer. The experimentally determined values of f_mα plotted against f_wα determined experimentally (■) or computed by AGADIR (○).