ChemBioChem 2003, 4, 1151 ± 1163 DOI: 10.1002/cbic.200300568 ¹ 2003 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 1151 4-Fluorophenylglycine as a Label for 19 F NMR Structure Analysis of Membrane-Associated Peptides Sergii Afonin, [a] RalfW. Glaser, [b] Marina Berditchevskaia, [c] Parvesh Wadhwani, [a] Karl-Heinz G¸hrs, [d] Ute Mˆllmann, [e] Andrea Perner, [e] and Anne S. Ulrich* [a, c] The non-natural amino acid 4-fluorophenylglycine (4F-Phg) was incorporated into several representative membrane-associated peptides for dual purpose. The 19 F-substituted ring is directly attached to the peptide backbone, so it not only provides a well- defined label for highly sensitive 19 F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F-Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide ™B18∫ were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F-Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic b-sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of D-4F-Phg reduced the biological activity of the peptide by interfering with its amphiphilic a-helical fold. Finally, to illustrate the numerous uses of L-4F-Phg in 19 F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid-state 19 F NMR spectroscopy of macroscopically oriented samples re- vealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide. Introduction Protein±lipid interactions are of fundamental relevance to cellular processes occurring at the plasma membrane and organelles. For example, antimicrobial peptides are used by many organisms as a first-line defence, as they are able to permeabilise the membranes of invading bacteria. [1] Fusogenic peptides represent the minimal membrane-perturbing part of various proteins that are involved in inter- and intracellular fusion processes. [2] Many different amphiphilic peptides and proteins interact permanently or reversibly with membranes, to operate as ion channels, transporters, toxins, enzymes, etc. As a functional picture of these systems is beginning to emerge, new experimental strategies are needed to study the structure and dynamics of peptides when they are bound to the lipid bilayer. Fluorescence and electron paramagnetic resonance spectros- copy are sensitive methods commonly used to characterise peptides in membranes. In many cases, however, bulky labels have to be incorporated, which may affect the peptide±lipid interactions studied. Depending on the mode of attachment, a flexible reporter group may not reflect the behaviour of the labelled molecule that well. Nonperturbing methods, such as solid-state NMR spectroscopy with selective 2 H, 13 Cor 15 N labels, on the other hand, are highly informative but are not very sensitive as they require comparatively large amounts of material. [3] Protons are, of course, the most sensitive reporters, but in view of their natural abundance it is not yet feasible to investigate membrane-bound peptides by solid-state 1 HNMR [a] Prof. Dr. A. S. Ulrich, S. Afonin, Dr. P. Wadhwani Forschungszentrum Karlsruhe, IFIA P.O.B. 3640, 76021 Karlsruhe (Germany) Fax.: ( 49) 721-608-4823 E-mail: anne.ulrich@ifia.fzk.de [b] Dr. R. W. Glaser Institute f¸r Biochemie und Biophysik Friedrich-Schiller-Universit‰t Jena Hans-Knˆll-Strasse 2, 07745 Jena (Germany) [c] Prof. Dr. A. S. Ulrich, Dr. M. Berditchevskaia Institut f¸r Organische Chemie, Universit‰t Karlsruhe Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany) [d] Dr. K.-H. G¸hrs Institut f¸r Molekulare Biotechnologie Beutenbergstrasse 11, 07745 Jena (Germany) [e] Dr. U. Mˆllmann, A. Perner Hans-Knˆll-Institut f¸r Naturstoff-Forschung e.V. Beutenbergstrasse 11a, 07745 Jena (Germany)