Journal of Molecular Biology
Folding Amphipathic Helices Into Membranes: Amphiphilicity Trumps Hydrophobicity
Introduction
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–non-polar asymmetry, characterized mathematically by the so-called hydrophobic moment1 (μH), 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 proteins2,3 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 complex.6 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 peptides.7., 8., 9., 10., 11., 12. Helix amphiphilicity has been widely examined in the context of membrane permeabilization, but little attention has been paid to the relationships between μH, peptide helix-forming ability, and membrane affinity. We present here the results of a systematic investigation of the influence of μH on the folding and partitioning of membrane-active peptides. We show that μH is a far more potent driving force for interfacial partitioning than total peptide hydrophobicity.
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 structure,13., 14., 15., 16. a process conveniently described as partitioning-folding coupling.17,18 A classic example is the partitioning of melittin, a 26-residue peptide that is the principal component of bee venom.19 Largely unstructured when free in solution, melittin strongly adopts an amphipathic α-helical conformation when partitioned into membranes.20., 21., 22., 23. An important driving force for folding arises from the lower energetic cost of partitioning H-bonded peptide bonds compared to free peptide bonds.17,18,23 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, ΔGresidue, 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 ΔGresidue for α-helical peptides range between −0.1 and −0.4 kcal mol−1 per residue. Ladokhin & White23 estimated that ΔGresidue = −0.41(±0.06) kcal mol−1 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-workers,25 using a variant of the native/diastereomeric approach, measured the partitioning of the antimicrobial peptide magainin into small unilamellar vesicles (SUV) formed from zwitterionic palmitoyloleoylphosphatidylcholine (POPC) and anionic palmitoyloleoylphosphatidylglycerol (POPG). They reported a value of only −0.14 kcal mol−1 per residue for ΔGresidue. Subsequently, Li et al.26 published a value of −0.25(±0.05) kcal mol−1 per residue, using model host-guest fusion peptides. We show here that such differences in ΔGresidue can arise in part from differences in μH.
Helical peptides are typically rendered amphipathic by using combinations of charged and hydrophobic residues.27 But for the experiments reported here, we wished to avoid charged residues because of the non-additivity of Coulombic and hydrophobic interactions,28 and because we wished to examine whether μH 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 phases.29 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 μH 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 μH, as does the magnitude of peptide partitioning free energy.
Section snippets
Thermodynamic framework
Algorithms for predicting peptide folding and binding to membrane interfaces require an experimentally accessible thermodynamic cycle for analyzing partitioning-folding data30,31 that yields ΔGresidue. Figure 2(a) shows the thermodynamic cycle that forms the quantitative framework for our data. Its important feature is an experimentally definable unfolded reference state in the aqueous phase, which, as discussed below, is critical for predictions. We consider an equilibrium between four states:
Discussion
We have examined the folding in water and membranes of a family of uncharged peptides of fixed amino acid composition (Ac-A8Q3L4-GW-NH2), designed to have different amphiphilicities as measured by the hydrophobic moment1 (μH). We have shown that all of the peptides form α-helical secondary structure in both water and membranes. Because all of the peptides have the same hydrophobicity, differences in folding and binding free energies must be due to structural differences described by μH. In
Materials
POPC and POPG were purchased from Avanti Polar Lipids (Alabaster, AL). Fmoc amino acids and resins for peptide synthesis were obtained from NovaBiochem (EMD Biosciences, San Diego, CA). All chemicals were of analytical reagent grade. A 10 mM potassium phosphate buffer solution (pH 7.0) was used to reduce the UV absorbance in CD experiments.
Peptide syntheses and purification
Peptides were synthesized on a 433A Applied Biosystems automatic synthesizer by step-wise solid-phase procedures51 using fluorenylmethoxycarbonyl (FMOC)
Acknowledgements
We thank Michael Myers for his editorial assistance and Drs Hirsh Nanda and Ryan Benz for providing coordinates from the simulation of melittin in a bilayer. This research was supported by grants to SHW and ASL from the National Institute of General Medical Sciences, US National Institutes of Health.
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Present address: M. Fernández-Vidal, Department of Peptide and Protein Chemistry, IIQAB-CSIC, 08034 Barcelona, Spain.