DOI: 10.1002/cphc.200700477 Conformation and Interaction of a d,l- Alternating Peptide with a Bilayer Membrane: X-ray Reflectivity, CD, and FTIR Spectroscopy** Andrea Küsel, [a] Ziad Khattari, [b] Philipp E. Schneggenburger, [a] Arnab Banerjee, [b] Tim Salditt,* [b] and Ulf Diederichsen* [a] Introduction A recently published X-ray structure of the peptide H-(Tyr-Tyr) 4 - Lys-OH (Tyr = d-Tyr) with an alternating configuration of amino acids shows a unique molecular network of right-handed anti- parallel double-stranded b 5.6 helices organized in peptide tubes. [1] Aggregation of these self-assembled peptide nano- tubes is enabled by hydrogen bonds between water molecules and the tyrosine side chains. X-ray structures obtained earlier by Lorenzi and co-workers through crystallization from organic nonpolar medium indicate that the phenylalanyl peptide Boc- (Phe-Phe) 4 -OMe (Boc = tert-butoxycarbonyl) forms a right- handed antiparallel b 5.6 helix. [2] Nevertheless, left-handed anti- parallel double-stranded b helices can also be obtained with aromatic or nonaromatic amino acids. [3,4] Several structural studies of d,l-alternating peptides under- line their potential to adopt different b-type conformations. [4–7] It has been demonstrated that parameters such as the number of residues per turn, pitch, and resulting diameter, as well as the handedness, can change under various external condi- tions. [8] Gramicidin A (gA), a natural monovalent antibiotic pen- tadecapeptide of d,l-alternating configuration from Bacillus brevis, [9,10] is found in different conformations depending on its environment. [11–18] Several studies on the structural behavior of gA and its analogues have been performed to understand their gating and transport mechanisms, [7,19–21] followed by a controversial discussion about the conducting form of gA. [8,17,19] Special attention has been given to peptide–lipid in- teractions, which are able to modulate the peptide structure and dynamics and, therefore, also its activity in lipid mem- branes. [22,23] Herein, we introduce novel synthetic analogues and a com- bination of biophysical techniques to elucidate the corre- sponding peptide–lipid interaction as a first step towards the rational design of artificial pore-forming peptides. The secon- dary structure of the presented peptide H-(Phe-Tyr) 5 -Trp-Trp- OH (1) reconstituted in different model membranes was inves- tigated by circular dichroism (CD) and Fourier-transform infra- red (FTIR) spectroscopy. In particular, we investigated the pep- tide structures in model bilayers of 1,2-dilauroylphosphatidyl- choline (DLPC) and 1,2-dimyristoylphosphatidylcholine (DMPC) multilamellar stacks as a function of peptide-to-lipid ratio (P/L) and the level of hydration. In addition, the effect of peptide in- corporation on the structure of the lipid bilayers is studied by X-ray reflectivity experiments. Structural techniques, such as X-ray scattering, provide direct information about the vertical density profile 1(z) of lipid [a] Dr. A. Küsel, P.E. Schneggenburger, Prof. Dr. U. Diederichsen Institut für Organische und Biomolekulare Chemie Georg-August-Universität Gçttingen Tammannstr. 2, 37077 Gçttingen (Germany) Fax:(+ 49)551-392944 E-mail:udieder@gwdg.de [b] Dr. Z. Khattari, + A. Banerjee, Prof. T. Salditt Institut für Rçntgenphysik, Georg-August-Universität Gçttingen Friedrich-Hund-Platz 1, 37077 Gçttingen (Germany) Fax:(+ 49)551-399430 E-mail:tsaldit@gwdg.de [ + ] Current address: Physics Department, Hashemite University, Zarqa (Jordan) [**] CD: Circular Dichroism, FTIR: Fourier-transform Infrared. Peptides with alternating amino acid configuration provide heli- cal secondary structures that are especially known from the membrane channel and pore-forming gramicidin A. In analogy to this natural d,l-alternating pentadecapeptide, the potential of d,l-alternating peptides for membrane insertion is investigated using the model dodecamer peptide H-(Phe-Tyr) 5 -Trp-Trp-OH. This aromatic peptide is introduced as a novel pore-forming syn- thetic analogue of gramicidin A. It forms a well-organized homo- dimer similar to one of the gramicidin A transmembrane motifs. X-ray reflectivity measurements are performed on solid-supported peptide–lipid complexes to obtain information about the influ- ence of the artificial dodecamer peptide on the bilayer parame- ters. In addition, Fourier-transform infrared (FTIR) and circular di- chroism (CD) spectroscopic studies determine the conformational state of H-(Phe-Tyr) 5 -Trp-Trp-OH within the model membrane. Site-specific iodine labeling assists in determining the topology of the membrane-embedded peptide by pinpointing the position of the iodine label within the bilayers. 2336 # 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemPhysChem 2007,8,2336–2343