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 seventeenresidue 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. Keywordsantimicrobial peptides; toxins; membrane proteins; peptide secondary structure; hydrophobic momentThe 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 proteins 2,3 where amphipathic helices apparently provide structural stability. But they are also important functionally. For example, amphipathic helices play important functional roles in both ligand-gated 4 ( Figure 1) and voltage-gated Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public AccessAuthor Manuscript J Mol Biol. Author manuscript; available in PMC 2008 July 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript K + channels 5 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 7-12 . Helix amphiphilicity has been ...

The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (Ln, n ‫؍‬ 4 … 12) flanked at either end by 4-residue sequences of the form XXPX-Ln-XPXX with X ‫؍‬ G, N, D, or K. Except for X ‫؍‬ K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n ‫؍‬ 12. For X ‫؍‬ K, p is already close to 100% for n ‫؍‬ 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.hydrophobic mismatch ͉ membrane protein synthesis ͉ membrane proteins ͉ molecular dynamics simulation ͉ lipid bilayer

Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the ''nonclassical'' hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (-TDS) and enthalpic (DH) components of free energy (DG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in DG (entropy-enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about -0.5 kcal mol -1 K -1 . This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy DG hU , which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543-552, 2001]. Our results suggest that there is no need for a special effect-the nonclassical hydrophobic effect-to describe partitioning into lipid bilayers.