Analogues of a synthetic ion channel made from a helical peptide were used to study the mechanism of cation translocation within bilayer membranes. Derivatives bearing two, three, four, and six crown ethers used as ion relays were synthesized, and their transport abilities across lipid bilayers were measured. The results showed that the maximum distance a sodium ion is permitted to travel between two binding sites within a lipid bilayer environment is 11 Å.

No abstract

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.

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.

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. (31)P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, (2)H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.

We present experimental data that demonstrate the potential of synthetic crown ether modified peptide nanostructures to act as selective and efficient chemotherapeutic agents that operate by attacking and destroying cell membranes.

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 channel's 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.

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 a-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 a-helical conformation and membrane incorporation of new nanostructures while 23 Na NMR assays shown a significant increase of their ion transport ability.