Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using ' H 2D NMR. This structure, involving an a-helix and a twisted three-stranded P-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized aP (CSaP) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short-and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.

Nonspecific lipid transfer protein from wheat is studied by liquid-state NMR in the presence of xenon. The gas-protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser-polarized xenon to the protein protons. Twenty-six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns-LTP cavity. Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by ∼12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.Keywords: Laser-polarized xenon; SPINOE; wheat nonspecific lipid transfer protein; protein hydrophobic cavity Supplemental material: See www.proteinscience.org.The catalytic sites of many enzymes are located in hydrophobic pockets. The content of these cavities in the absence of substrate is still subject to debate because the presence of water molecules may play a fundamental role in the thermodynamics of the enzymatic activity (Ernst et al. 1995;Matthews et al. 1995;Quillin et al. 2000). Currently, only two physical methods allow direct characterization of these hydrophobic cavities at the atomic level: i) X-ray diffraction of a protein crystal under noble gas pressure (xenon, krypton,. . .; Montet et al. 1997;Prangé et al. 1998;Quillin et al. 2000). The disadvantages of this method are that mediumto-high pressures must be used, and the dynamic properties of the interaction cannot easily be understood; ii) hydration studies using liquid-state 1 H NMR, which involve solventprotein protons cross-relaxation. These measurements can be delicate because exchange between water and hydroxyl protons can mask the through-space interactions between the solvent and the protein protons (Otting et al. 1991). A variation of this method is the use of small organic molecules that can probe cavities. Then the dependence of the internuclear distances on the intermolecular cross-relaxation rates between the protons of the protein and of the organic compound can be exploited (Otting et al. 1997). However, as these nOe signals are usually proportional to the concentration of the small organic compounds (fast-exchange conditions), improving the sensitivity of this approach requires very high pressures (e.g., 200 bars of methane). This may induce structural modifications of the protein (Prangé et al.Reprint requests to: Dr. Hervé Desvaux, Service de Chimie Moléculaire, CEA/Saclay, F-91191 Gif sur Yvette, France; e-mail: hdesvaux@Cea.fr; fax: 33-1-69-08-98-06.Abbreviations: FID, Free Induction Decay; Ns-LTP, nonspecific lipid transfer protein; nOe, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; SPI-NOE, spin polarization-...

In response to an experimental infection, the lepidopteran Heliothis virescens produces an antifungal protein named heliomicin. Heliomicin displays sequence similarities with antifungal plant defensins and antibacterial or antifungal insect defensins. To gain information about the structural elements required for either antifungal or antibacterial activity, heliomicin and selected point-mutated variants were expressed in yeast as fusion proteins. The effects of mutations, defined by comparing the primary structure of heliomicin with the sequences of members of the insect defensin family, were analyzed using antibacterial and antifungal assays. One of the variants shows significant activity against Gram-positive bacteria while remaining efficient against fungi. The three-dimensional structures of this variant and of the wild-type protein were determined by two-dimensional (1)H NMR to establish a correlation between structure and antibacterial or antifungal activity. Wild-type and mutated heliomicins adopt a similar scaffold, including the so-called cysteine-stabilized alphabeta motif. A comparison of their structures with other defensin-type molecules indicates that common hydrophobic characteristics can be assigned to all the antifungal proteins. A comparative analysis of various structural features of heliomicin mutant and of antibacterial defensins enables common properties to be assessed, which will help to design new mutants with increased antibacterial activity.