Solution structure of Ace-AMP1, a potent antimicrobial protein extracted from onion seeds. Structural analogies with plant nonspecific lipid transfer proteins.

The three-dimensional solution structure of Ace-AMP1, an antifungal protein extracted from onion seeds, was determined using 1H NMR spectroscopy and molecular modeling. This cationic protein contains 93 amino acid residues and four disulfide bridges. Its structure was determined from 1260 NOE-derived distance restraints and 173 dihedral restraints derived from NOEs and 3JCaHNH coupling constants. The global fold involves four helical segments connected by three loops and a C-terminal tail without regular secondary structures, except for a 3(10)-helix turn and a beta-turn. The most striking feature is the absence of any continuous cavity running through the whole molecule as found in recently determined structures of nonspecific transfer proteins extracted from wheat and maize seeds, although their global folds are very similar. Consistent with the absence of a cavity in the core of Ace-AMP1, it was found that this protein, in contrast to ns-LTPs, does not bind fluorescently labeled phospholipids in solution. On the other hand, Ace-AMP1 is able to interact with phospholipid membranes as shown by the release of carboxyfluorescein from the lumen of artificial liposomes and by the induction of alterations in fluorescence polarization of fluorescently labeled phospholipids embedded in artificial liposomes.

Lipopeptides with improved properties: structure by NMR, purification by HPLC and structure-activity relationships of new isoleucyl-rich surfactins.

The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L-isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified by beta-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure-activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed.

Structure of the intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex: I. A 1H and 31P n.m.r. study.

The structure of an intrastrand cis-[Pt(NH3)2(d(GpCpG))] adduct in a dodecanucleotide duplex has been investigated by using ultraviolet absorption, circular dichroism, 1H and 31P n.m.r. The binding of cis-DDP does not inhibit the formation of a duplex but it induces a lowering of congruent to 26 degrees C of its melting temperature. A broadening of the 1H spectrum prevents an accurate analysis of the platination site. Nevertheless, by considering its thermal behavior and the number of imino protons a model of structure of the platinated duplex is proposed in which the central C.G. pair is disrupted and a neighboring C.G pair is very accessible or distorted. The environment of two phosphate groups is disturbed by the cis-DDP binding.