FOLDING AMPHIPATHIC HELICES INTO MEMBRANES: AMPHIPHILICITY TRUMPS HYDROPHOBICITY

Mónica Fernández-Vidal

Sajith Jayasinghe

Alexey Ladokhin

Stephen White

^{Department of Physiology and Biophysics, University of California at Irvine, Irvine, CA 92697-4560, USA}

^{Department of Chemistry and Biochemistry, California State University at San Marcos, San Marcos, CA 92096, USA}

^{Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421, USA}

Correspondence should be addressed to S.H.W. (ude.icu@etihw.nehpets) or A.S.L. (ude.cmuk@nihkodala).

^{Present address: Dept. of Peptide and Protein Chemistry, IIQAB-CSIC, 08034 Barcelona, Spain.}

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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 seventeen-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.

Keywords: antimicrobial peptides, toxins, membrane proteins, peptide secondary structure, hydrophobic moment

Abstract

The amphipathic (or amphiphilic) helix is an important structural motif in proteins. Its most common representation shows polar residues along the length of one-half of a helix surface and non-polar residues along the opposite surface (Figure 1). This polar-nonpolar asymmetry, characterized mathematically by the so-called hydrophobic moment1 (μ_H), makes the amphipathic helix ideally suited for binding to membrane interfaces with the polar surface facing the aqueous phase and the less polar surface facing the membrane interior. This arrangement is often seen in membrane proteins²3 where amphipathic helices apparently provide structural stability. But they are also important functionally. For example, amphipathic helices play important functional roles in both ligand-gated⁴ (Figure 1) and voltage-gated K^channels⁵ and in the insertion of disulfide bonds into Escherichia coli periplasmic proteins by the DsbB-DsbA complex⁶. Because of its tendency to partition into membrane interfaces (Figure 1) and subsequently permeabilize membranes, the amphipathic helix is a common starting motif for designing or re-engineering antimicrobial peptides⁷12. Helix amphiphilicity has been widely examined in the context of membrane permeabilization, but little attention has been paid to the relationships between μ_H, peptide helix-forming ability, and membrane affinity. We present here the results of a systematic investigation of the influence of μ_H on the folding and partitioning of membrane-active peptides. We show that μ_H is a far more potent driving force for interfacial partitioning than total peptide hydrophobicity.

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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 simulation⁵⁵. (b) Channel domain of the KirBac1.1 ligand-gated K^{-channel in the closed state}⁴, 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 proteins²3. Images produced with VMD software⁵⁶.

Most membrane-active helix-forming peptides have low or moderate helicity in aqueous solution but become highly helical when partitioned into membranes. This is due in part to the potent ability of membranes to promote secondary structure¹³16, a process conveniently described as partitioning-folding coupling¹⁷18. A classic example is the partitioning of melittin, a 26-residue peptide that is the principal component of bee venom¹⁹. Largely unstructured when free in solution, melittin strongly adopts an amphipathic α-helical conformation when partitioned into membranes²⁰23. An important driving force for folding arises from the lower energetic cost of partitioning H-bonded peptide bonds compared to free peptide bonds¹⁷1823. Knowledge of the energetics of this folding process is important for improving the activity of antimicrobial peptides and for understanding the folding and stability of membrane proteins. An essential element of these energetics is the per-residue reduction in free energy, ΔG_residue, that drives secondary structure formation in the membrane interface. This parameter, as we shall show, plays a critical role in the development of an analytical description of peptide folding.

Reported values for ΔG_residue for α-helical peptides range between −0.1 and −0.4 kcal mol^{per residue. Ladokhin and White}²³ estimated that ΔG_residue = −0.41(±0.06) kcal mol^{for melittin partitioning-folding in zwitterionic large unilamellar vesicles (LUV) by measuring the partitioning free energies and helicities of native melittin and of a diastereomeric analog with four D-amino acids (D4, L-melittin)}²⁴. At about the same time, Seelig and co-workers²⁵, using a variant of the native/diastereomeric approach, measured the partitioning of the antimicrobial peptide magainin into small unilamellar vesicles (SUV) formed from POPC and anionic palmitoyloleoylphosphatidylglycerol (POPG). They reported a value of only −0.14 kcal mol^{per residue for Δ}G_residue. Subsequently, Li et al.²⁶ published a value of −0.25(±0.05) kcal mol^{per residue, using model host-guest fusion peptides. We show here that such differences in Δ}G_residue can arise in part from differences in μ_H.

Helical peptides are typically rendered amphipathic by using combinations of charged and hydrophobic residues²⁷. But for the experiments reported here, we wished to avoid charged residues because of the non-additivity of Coulombic and hydrophobic interactions²⁸, and because we wished to examine whether μ_H effects are affected by surface charge. We therefore used electrically neutral peptides whose designs were inspired by the peptides that Baldwin and colleagues used for studies of α-helix stability in aqueous phases²⁹. As we describe below, we synthesized a family of peptides of the general form Ac-A₈Q₃L₄-GW-NH₂ in which we varied the A₈Q₃L₄ sequence to cover a range of μ_H values. The result was a family of peptides with identical hydrophobicities but different hydrophobic moments. We report below each peptide’s helicity and folding free energy in buffer and in POPC and POPC:POPG LUV. We show that peptide helicity in water and interface increase linearly with μ_H, as does the magnitude of peptide partitioning free energy.

Footnotes

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Footnotes

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