The use of AFM to study molecular recognition events at an incredible level of sensitivity is currently a very active field of research. In order to get information at the single molecule level, it is mandatory to modify in a precise manner the AFM tip to anchor either ligand or receptor molecules. In the following lines, we review the achievements in tip modifications and illustrate the scope and limitations of the different strategies that have been reported.

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.

A helical 14-residue peptide containing four polar, but uncharged, benzo-21-crown-7 side-chains aligned along one face induces significantly more vesicle leakage than analogous 21-mer or 7-mer peptides.

Ion channel proteins are complex membrane proteins that control and regulate the transport of ions across cell membranes. Because of their involvement in several diseases,' their potential use in biosensors? and their inherent difficulty of isolation, intense efforts have been devoted to the preparation of artificial ion channel^.^ Here, we report a very simple, rapid, and efficient strategy for the preparation of such functional channel molecules. The strategy combines the versatility of solid phase peptide synthesis, the conformational predictability of peptidic molecules, and the solution synthesis of crown ethers with engineerable ion-binding abilities.

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tains 15 L-leucine residues it is both sufficiently hydrophobic to be incorporated in lipid bilayers[41 and favors u-helical conformation. ['] In this form the crown ether rings in I are perfectly aligned (Figure 1). Herein we present the results of studies on the conformation of 1 and its incorporation in planar C.

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.

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNAmediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2,4-diacetoxy-5-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.