Selective fluorination of peptides results in increased chemical and thermal stability with simultaneously enhanced hydrophobicity. We demonstrate here that fluorinated derivatives of two host defense antimicrobial peptides, buforin and magainin, display moderately better protease stability while retaining, or exhibiting significantly increased bacteriostatic activity. Four fluorinated analogues in the buforin and two in the magainin series were prepared and analyzed for (1) their ability to resist hydrolytic cleavage by trypsin; (2) their antimicrobial activity against both gram-positive and gram-negative bacterial strains; and (3) their hemolytic activity. All but one fluorinated peptide (M2F5) showed retention, or significant enhancement, of antimicrobial activity. The peptides also showed modest increases in protease resistance, relative to the parent peptides. Only one of the six fluorinated peptides (BII1F2) was degraded by trypsin at a slightly faster rate than the parent peptide. Hemolytic activity of peptides in the buforin series was essentially null, while fluorinated magainin analogues displayed an increase in hemolysis compared to the parent peptides. These results suggest that fluorination may be an effective strategy to increase the stability of biologically active peptides where proteolytic degradation limits therapeutic value.

Selective incorporation of unnatural amino acids into proteins is a powerful tool for illuminating the principles of protein design. In particular, fluorinated amino acids have recently emerged as valuable building blocks for designing hyperstable protein folds, as well as directing highly specific protein-protein interactions. We review the collagen mimetic and coiled coil peptide systems that exemplify generalizable paradigms for future design. The unique electronic and phase properties of fluorocarbons are discussed, and protein synthesis using unnatural amino acids is briefly reviewed.

Specific protein-protein interactions are crucial for virtually all biological processes. 1 Naturally occurring surfaces that mediate protein-protein interactions usually contain either elements of polar specificity, for example hydrogen bonding or salt bridges, or complementary hydrophobic patches that contain side chains that maximize van der Waals interactions. 2 We, 3 and others 4 have recently described a new type of protein-protein interaction interface that could potentially be both hydrophobic and lipophobic. This is accomplished by the introduction of nonproteinogenic, fluorine-containing amino acids. 5,6 Reported here is the design, synthesis, and programmed self-sorting of peptide systems with orthogonally miscible hydrocarbon and fluorous (highly fluorinated) cores.Peptides H and F are equipped with N-terminal cysteine residues and were designed to form parallel homodimeric coiled coil assemblies. 7 These peptides have an identical sequence except that all seven of the core leucine residues in H have been replaced by 5,5,5,5′,5′,5′-R-S-hexafluoroleucine in F (Figure 1), shielding 28 trifluoromethyl groups from aqueous solvent in the canonical fluorinated dimer. Hexafluoroleucine was synthesized according to a recently documented procedure. 8 The peptides were assembled on 4-methylbenzhydrylamine (MBHA) resin according to the in situ neutralization protocol for t-Boc peptide synthesis as described previously 9 and purified by reverse-phase HPLC. Purity of the peptides was confirmed by analytical HPLC and MALDI mass spectrometry. H and F are designed to form parallel coiled coil structures due to unfavorable interhelical electrostatic interactions in the antiparallel arrangements. 10 Furthermore, a single polar residue Asn14 was incorporated in the hydrophobic

The design, synthesis, and structural characterization of a highly fluorinated peptide system based on the coiled coil region of the yeast transcription factor GCN4 is described. All four leucine residues (a position) and three valine residues (d position) were replaced by the unnatural amino acids 5,5,5-trifluoroleucine and 4,4,4-trifluorovaline, respectively. The peptide is highly alpha-helical at low micromolar concentrations as judged by circular dichroism spectra, sediments as a dimeric species in the 5-30 microM concentration range, and exhibits a dimer melting temperature that is 15 degrees C higher than a control peptide with a hydrocarbon core. Furthermore, the apparent free energy of unfolding as calculated from guanidinium hydrochloride denaturation experiments is larger by approximately 1.0 kcal/mol for the fluorinated peptide than its hydrocarbon counterpart. We conclude that additional stability is derived from sequestering the more hydrophobic trifluoromethyl groups from aqueous solvent. These studies introduce a new paradigm in the design of molecular self-assembling systems, one based on orthogonal solubility properties of liquid phases.

Control of structure and function in membrane proteins remains a formidable challenge. We report here a new design paradigm for the self-assembly of protein components in the context of nonpolar environments of biological membranes. An incrementally staged assembly process relying on the unique properties of fluorinated amino acids was used to drive transmembrane helixhelix interactions. In the first step, hydrophobic peptides partitioned into micellar lipids. Subsequent phase separation of simultaneously hydrophobic and lipophobic fluorinated helical surfaces fueled spontaneous self-assembly of higher order oligomers. The creation of these ordered transmembrane protein ensembles is supported by gel electrophoresis, circular dichroism spectroscopy, equilibrium analytical ultracentrifugation, and fluorescence resonance energy transfer.

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The characterization of the lateral organization of components in biological membranes and the evolution of this arrangement in response to external triggers remains a major challenge. The concept of lipid rafts is widely invoked, however, direct evidence of the existence of these ephemeral entities remains elusive. We report here the use of Secondary Ion Mass Spectrometry (SIMS) to image the cholesterol-dependent cohesive phase separation of the ganglioside GM1 into nano and micro-scale assemblies in a canonical lipid raft composition of lipids. This assembly of domains was interrogated in a model membrane system composed of palmitoyl sphingomyelin (PSM), cholesterol, and an unsaturated lipid (dioleoylphosphatidylcholine, DOPC). Orthogonal isotopic labeling of every lipid bilayer component and monofluorination of GM1 allowed generation of molecule specific images using a NanoSIMS. Simultaneous detection of six different ion species in SIMS, including secondary electrons, was used to generate ion ratio images whose signal intensity values could be correlated to composition through the use of calibration curves from standard samples. Images of this system provide the first direct, molecule specific, visual evidence for the co-localization of cholesterol and GM1 in supported lipid bilayers and further indicate the presence of three compositionally distinct phases: (1) the interdomain region; (2) micrometer-scale domains (d>3 μm); and, (3) nanometer-scale domains (d=100 nm − 1 μm) localized within the micrometer-scale domains and the interdomain region. PSM-rich, nanometer-scale domains prefer to partition within the more ordered, cholesterol-rich/DOPC-poor/GM1-rich micrometer-scale phase, while GM1-rich, nanometer-scale domains prefer to partition within the surrounding, disordered, cholesterol-poor/PSM-rich/DOPC-rich interdomain phase.

The multivalent carbohydrate-carbohydrate interaction between membrane anchored epitopes derived from the marine sponge Microciona prolifera (M. prolifera) has been explored by colloidal probe microscopy. An in situ coupling of sulfated and non-sulfated disaccharides to membrane coated surfaces was employed to mimic native cell-cell contacts. The dynamic strength of the homomeric self-association was measured as a function of calcium ion concentration and loading rate. A deterministic model was used to estimate the number of participating bonds in the contact zone.