François Otis

Exploiting Peptide Nanostructures To Construct Functional Artificial Ion Channels

Natural ion channel proteins possess remarkable properties that researchers could exploit to develop nanochemotherapeutics and diagnostic devices. Unfortunately, the poor stability, limited availability, and complexity of these structures have precluded their use in practical devices. One solution to these limitations is to develop simpler molecular systems through chemical synthesis that mimic the salient properties of artificial ion channels. Inspired by natural channel proteins, our group has developed a family of peptide nanostructures thatcreate channels for ions by aligning crown ethers on top of each other when they adopt an α-helical conformation. Advantages to this crown ether/peptide framework approach include the ease of synthesis, the predictability of their conformations, and the ability to fine-tune and engineer their properties. We have synthesized these structures using solid phase methods from artificial crown ether amino acids made from L-DOPA. Circular dichroism and FTIR spectroscopy studies in different media confirmed that the nanostructures adopt the predicted α-helical conformation. Fluorescence studies verified the crown ether stacking arrangement. We confirmed the channel activity by single-channel measurements using a modified patch-clamp technique, planar lipid bilayer (PLB) assays, and various vesicle experiments. From the results, we estimate that a 6 Å distance between two relays is ideal for sodium cation transport, but relatively efficient ion transport can still occur with an 11 Å distance between two crown ethers. Biophysical studies demonstrated that peptide channels operate as monomers in an equilibrium between adsorption at the surface and an active, transmembrane orientation. Toward practical applications of these systems, we have prepared channel analogs that bear a biotin moiety, and we have used them as nanotransducers successfully to detect avidin. Analogs of channel peptide nanostructures showed cytotoxicity against breast and leukemia cancer cells. Overall, we have prepared well-defined nanostructures with designed properties, demonstrated their transport abilities, and described their mechanism of action. We have also illustrated the advantages and the versatility of polypeptides for the construction of functional nanoscale artificial ion channels.

End group engineering of artificial ion channels

Toward developing nanostructures for single molecule detection, we report the synthesis of new derivatives of artificial devices that possess ion channel activity. The membrane stability and the ion transport ability of these multiple crown α-helical peptides have been optimized by modulating the polarity of N- and C-terminal groups with incorporation of hydrophilic non-ionic units. Circular dichroism and ATR studies confirmed the α-helical conformation and membrane incorporation of new nanostructures while 23Na NMR assays shown a significant increase of their ion transport ability.

Membrane Assembly and Ion Transport Ability of a Fluorinated Nanopore.

A novel 21-residue peptide incorporating six fluorinated amino acids was prepared. It was designed to fold into an amphiphilic alpha helical structure of nanoscale length with one hydrophobic face and one fluorinated face. The formation of a fluorous interface serves as the main vector for the formation of a superstructure in a bilayer membrane. Fluorescence assays showed this ion channels ability to facilitate the translocation of alkali metal ions through a phospholipid membrane, with selectivity for sodium ions. Computational studies showed that a tetramer structure is the most probable and stable supramolecular assembly for the active ion channel structure. The results illustrate the possibility of exploiting multiple Fδ-:M+ interactions for ion transport and using fluorous interfaces to create functional nanostructures.

Crown ether helical peptides are preferentially inserted in lipid bilayers as a transmembrane ion channels

Oriented circular dichroism was used to study the alignment crown ether‐modified peptides. The influence of different N‐ and C‐functionalities was assessed using at variable peptide:lipid ratios from 1:20 to 1:200. Neither the functionalities nor the concentration had any major effect on the orientation. The alignment of the 21‐mer peptides was also examined with lipid membranes of different bilayer thickness. The use of synchrotron radiation as light source allowed the study of peptide:lipid molar ratios from 1:20 to 1:1000. For all conditions studied, the peptides were found to be predominantly incorporated as a transmembrane helix into the membrane, especially at low peptide concentration, but started to aggregate on the membrane surface at higher peptide:lipid ratios. The structural information on the preferred trans‐bilayer alignment of the crown ether functional groups explains their ion conductivity and is useful for the further development of membrane‐active nanochemotherapeutics.

Characterization of channel-forming peptide nanostructures

We have prepared fluorescent analogs of known ion-channel-forming synthetic peptide nanostructures. These analogs were designed as probes to gain insight about the mechanism by which self-assembling amphiphilic peptides interact with lipid membranes. Conformational studies demonstrated that the labeled analogs retain their propensity to adopt a strong helical conformation in 2,2,2-trifluoroethanol and lipid bilayers. Attenuated total reflectance results indicated that the fluorescent peptide nanostructures are under an incorporation equilibrium between two forms, adsorbed at the surface or incorporated within the bilayer, similar to their unlabeled counterparts. However, when using a HeLa mimicking membrane, the proportion of peptide nanostructures in the transmembrane orientation decreases significantly. Finally, we were able to show by confocal microscopy studies that fluorescent analogs internalized into HeLa cells and localized into both the membranes of inner organelles and the cell membrane.

Synthesis and characterization of peptide nanostructures designed for sensing applications

We report the design and the synthesis of membrane-active peptide nanostructures, as well as their use as signal transducer in a fluorimetric assay for biologically relevant analytes. Addition of hydrophobic 21-residue peptides bearing six crown ether side chains to a solution of small unilamellar vesicles loaded with a pH-sensitive fluorophore induces a rapid fluorescence increase associated with Na+/H+ transport across the bilayer membrane. To demonstrate the usefulness of these peptide nanostructures in the development of simple, rapid, and sensitive detection assays for a wide range of analytes, peptide nanostructures bearing a biotin at the N-terminal position were prepared. Addition of avidin to the assay employing these modified peptides resulted in a significant change in the time-dependent fluorescence profile. Control experiments with a non-binding proteins and saturated avidin showed that the observed changes are indeed due to specific binding of avidin to biotin modified nanostructures.

Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Lipid membrane interactions of a fluorinated peptide with potential ion channel-forming ability

Fluorinated peptides attract much interest in the biomedical area because they generally exhibit an enhanced stability compared to their hydrogenated counterparts and because fluorine atoms represent efficient probes to investigate peptide assemblies, especially in membranes. We previously designed and characterized a fluorinated peptide intended to form ion channels in membranes. This peptide, designated as LX2, adopts a predominantly α‐helical structure and acts as a selective ion channel for various cations. Molecular dynamics indicated that the peptide tetrameric form would be favored. However, the interactions of LX2 with model membranes have not been studied experimentally. Here we investigated the interactions of LX2 and its acetylated form, Ac‐LX2, with eukaryotic model membranes using CD and infrared spectroscopy, as well as 19F, 2H, and 31P NMR spectroscopy. 19F NMR results indicate that the peptides undergo restrained motions in the presence of membranes, which strongly suggests their insertion in membranes. Both LX2 and Ac‐LX2 adopt a well‐defined α‐helical structure, in contrast with the secondary structure observed in hexafluoroisopropanol. The alterations of lipid organization due to LX2 peptides appear overall relatively small but the results suggest that the two peptides are located in the membrane hydrophobic core, LX2 being closer to the interfacial region than Ac‐LX2.

Inhibition of Helicobacter pylori Glu‐tRNAGln amidotransferase by novel analogues of the putative transamidation intermediate

Glutaminyl‐tRNAGln in Helicobacter pylori is formed by an indirect route requiring a noncanonical glutamyl‐tRNA synthetase and a tRNA‐dependent heterotrimeric amidotransferase (AdT) GatCAB. Widespread use of this pathway among prominent human pathogens, and its absence in the mammalian cytoplasm, identify AdT as a target for the development of antimicrobial agents. We present here the inhibitory properties of three dipeptide‐like sulfone‐containing compounds analogous to the transamidation intermediates, which are competitive inhibitors of AdT with respect to Glu‐tRNAGln. Molecular docking revealed that AdT inhibition by these compounds depends on π–π stacking interactions between their aromatic groups and Tyr81 of the GatB subunit. The properties of these inhibitors indicate that the 3′‐terminal adenine of Glu‐tRNAGln plays a major role in binding to the AdT transamidation active site.