Sergii Afonin

4-fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides

The non‐natural amino acid 4‐fluorophenylglycine (4F‐Phg) was incorporated into several representative membrane‐associated peptides for dual purpose. The 19F‐substituted ring is directly attached to the peptide backbone, so it not only provides a well‐defined label for highly sensitive 19F 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 β‐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 α‐helical fold. Finally, to illustrate the numerous uses of l‐4F‐Phg in 19F 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 19F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide.

Peptoidic Amino- and Guanidinium-Carrier Systems: Targeted Drug Delivery into the Cell Cytosol or the Nucleus

Efficient drug delivery is essential for many therapeutic applications. Some cell-penetrating peptides, peptide mimetics, and peptoids express transport function that, however, lack in most cases specific intracellular destination. In this study, carrier-peptoids with either amino or guanidinium side chains, were investigated with regard to their cellular uptake, toxicity, and intracellular localization. Transport specifically to the cytosol or to the nuclei was observed, thus providing a powerful tool for targeted drug delivery.

Conformation and Membrane Orientation of Amphiphilic Helical Peptides by Oriented Circular Dichroism

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic alpha-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of approximately 1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L approximately 1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L approximately 1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with beta-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.

Temperature-dependent transmembrane insertion of the amphiphilic peptide PGLa in lipid bilayers observed by solid state 19F NMR spectroscopy.

The alignment of the antimicrobial peptide PGLa in a lipid bilayer was characterized by solid state 19F NMR on selectively CF3-labeled peptides in oriented samples. Previous studies in liquid crystalline model membranes had shown that the amphiphilic α-helix of PGLa adopts a surface-aligned S-state or a tilted T-state, depending on the peptide/lipid ratio. Only in the presence of the synergistic partner peptide magainin 2 had PGLa been found insert into the bilayer in a transmembrane I-state orientation. Here, we have characterized the peptide alignment but as a function of temperature and lipid phase state. At temperatures below the lipid chain melting transition, PGLa is now seen to be able to insert on its own, with its helical axis nearly parallel to the bilayer normal (tilt angle of ∼170°), forming the I-state. Above the lipid phase transition, PGLa is aligned in the known T-state (tilt angle of ∼130°), but it is found flip into the S-state at more elevated temperatures (tilt angle of ∼96°). This way...

Controlling biological activity with light: diarylethene-containing cyclic peptidomimetics.

Photobiological processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analogue was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analogue was incorporated into the cyclic backbone of the antimicrobial peptide gramicidin S at several sites. The biological activity of the resulting peptidomimetics could then be effectively controlled by ultraviolet/visible light within strictly defined spatial and temporal limits.

Solid-State 19 F-NMR of Peptides in Native Membranes

To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues.Here, we discuss a solid-state (19)F-NMR approach that has been developed for structural studies of MAPs in lipid bilayers, and how this can be translated to measurements in native biomembranes. We review the essentials of the methodology and discuss key objectives in the practice of (19)F-labelling of peptides. Furthermore, the preparation of macroscopically oriented biomembranes on solid supports is discussed in the context of other membrane models. Two native biomembrane systems are presented as examples: human erythrocyte ghosts as representatives of eukaryotic cell membranes, and protoplasts from Micrococcus luteus as membranes from Gram-positive bacteria. Based on our latest experimental experience with the antimicrobial peptide gramicidin S, the benefits and some implicit drawbacks of using such supported native membranes in solid-state (19)F-NMR analysis are discussed.

19F NMR analysis of the antimicrobial peptide PGLa bound to native cell membranes from bacterial protoplasts and human erythrocytes.

(19)F NMR is a unique tool to examine the structure of fluorine-labeled peptides in their native cellular environment, due to an exquisite sensitivity and lack of natural abundance background. For solid-state NMR analysis, we isolated native membranes from erythrocyte ghosts and bacterial protoplasts and prepared them as macroscopically oriented samples. They showed a high purity and quality of alignment according to (31)P NMR, and the membrane-bound antimicrobial peptide PGLa could be detected by (19)F NMR. The characteristic fingerprint splitting of its (19)F reporter group indicated that the peptide helix binds to the native membranes in a surface alignment, albeit with a higher affinity in the prokaryotic than the eukaryotic system.

Hydrophobic Matching Controls the Tilt and Stability of the Dimeric Platelet-derived Growth Factor Receptor (PDGFR) β Transmembrane Segment

Background: Dimerization regulates activation of PDGF receptor in signal transduction. Results: The transmembrane segment of PDGFR forms a left-handed helical dimer, which becomes more tilted and less stable in model membranes with decreasing lipid acyl chain lengths. Conclusion: The membrane thickness controls the ability of the transmembrane segments to dimerize. Significance: Receptor dimerization and activation in vivo may require relocation to thick lipid rafts. The platelet-derived growth factor receptor β is a member of the cell surface receptor tyrosine kinase family and dimerizes upon activation. We determined the structure of the transmembrane segment in dodecylphosphocholine micelles by liquid-state NMR and found that it forms a stable left-handed helical dimer. Solid-state NMR and oriented circular dichroism were used to measure the tilt angle of the helical segments in macroscopically aligned model membranes with different acyl chain lengths. Both methods showed that decreasing bilayer thickness (DEPC-POPC-DMPC) led to an increase in the helix tilt angle from 10° to 30° with respect to the bilayer normal. At the same time, reconstitution of the comparatively long hydrophobic segment became less effective, eventually resulting in complete protein aggregation in the short-chain lipid DLPC. Unrestrained molecular dynamics simulations of the dimer were carried out in explicit lipid bilayers (DEPC, POPC, DMPC, sphingomyelin), confirming the observed dependence of the helix tilt angle on bilayer thickness. Notably, molecular dynamics revealed that the left-handed dimer gets tilted en bloc, whereas conformational transitions to alternative (e.g. right-handed dimeric) states were not supported. The experimental data along with the simulation results demonstrate a pronounced interplay between the platelet-directed growth factor receptor β transmembrane segment and the bilayer thickness. The effect of hydrophobic mismatch might play a key role in the redistribution and activation of the receptor within different lipid microdomains of the plasma membrane in vivo.

Chemical Labeling Strategy with (R)- and (S)-Trifluoromethylalanine for Solid State 19F NMR Analysis of Peptaibols in Membranes

Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints theta by solid state (19)F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles tau and rho from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an alpha-helix, tilted away from the membrane normal by 8 degrees, and assembled as an oligomer. The new (19)F NMR label CF(3)-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

Dynamic Transitions of Membrane-Active Peptides

Membrane-active peptides or protein segments play an important role in many biological processes at the cellular interface to the environment. They are involved, e.g., in cellular fusion or host defense, where they can cause not only merging but also the destabilization of cell membranes. Many factors determine how these typically amphipathic peptides interact with the lipid bilayer. For example, the peptide orientation in the membrane determines which parts of the peptide are exposed to the hydrophobic bilayer interior or to the polar lipid/water interface. As another example, oligomerization is required for many activities such as pore formation. Peptides have been often classified according to a single characteristic mode of interaction with the bilayer, but over the years a more versatile picture has emerged. It appears that any single peptide can adopt several different alignments and/or oligomeric states in response to changes in the environment. For instance, many antimicrobial peptides adopt a surface-parallel alignment at low concentration, but they tilt obliquely into or even fully insert transmembrane into the bilayer above a critical peptide-to-lipid ratio, often in the form of oligomeric pores. Similar changes in peptide orientation or oligomeric state have been observed as a function of, e.g., temperature, lipid composition, pH, or induced by a synergistic partner peptide. Such transitions between peptide states can be regarded as the result of a re-adjustment in the balance between peptide-peptide and peptide-lipid interactions, as the environment conditions are changed. Though often studied in model membrane systems, such rich variety of peptide states is even more likely to occur in native biomembranes with their diverse compositions and physicochemical properties. The ability to undergo transitions between different states thus plays a fundamental role for the biological activities of membrane-active peptides.