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Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI ␤-turn connecting ␤-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting ␤-strand 1 and the ␣-helix and contiguous residues on the ␣-helix and ␤-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca 2؉ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca 2؉ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca 2؉ uptake.

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1 H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69-74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.

The disulfide bridge closed cyclic peptide corresponding to the whole Consensus V3 loop of the envelope protein gpl20 of HIV-1 was examined by proton 2D-NMR spectroscopy in water and in a 20% trifluoroethanol/water solution. In water, NOE data support a B-turn conformation for the central conservative GPGR region and point towards partial formation of a helix in the C-terminal part. Upon addition of trifluoroethanol, a C-terminal helix is formed. This is evidenced by NOE data, a-proton chemical shift changes and changes in the JNo vicinal coupling constants. The C-terminal helix is amphipathic and also occurs in other examined strains. It could therefore be an important feature for the functioning of the V3 loop.

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