The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by ' H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (4,$, x,) derived from NOES and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for a-class proteins. 'H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, K d , of 1.9 f 0.6 x M and M were obtained with lyso-C,, and -Clz, respectively. A structure model for a lipidprotein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipidprotein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).

The antigenic activity of a 19-mer peptide corresponding to the major antigenic region of foot-and-mouth disease virus and its retro-enantiomeric analogue was found to be completely abolished when they were tested in a biosensor system in trifluoroethanol. This suggests that the folding pattern, which is ␣-helix in trifluoroethanol (confirmed by CD measurement), does not correspond to the biologically relevant conformation(s) recognized by antibodies. The NMR structures of both peptides were thus determined in aqueous solution. These studies showed that the two peptides exhibit similar folding features, particularly in their C termini. This may explain in part the cross-reactive properties of the two peptides in aqueous solution. However, the retro-inverso analogue appears to be more rigid than the parent peptide and contains five atypical ␤-turns. This feature may explain why retro-inverso foot-andmouth disease virus peptides are often better recognized than the parent peptide by anti-virion antibodies.The major immunogenic site of foot-and-mouth disease virus (FMDV), 1 which is contained in the so-called G-H loop comprising amino acid residues 135-158 of capsid protein VP1 (viral capsid protein 1), is located in a disordered region on the surface of the particle (1). A single inoculum of a peptide corresponding to this region usually elicits levels of neutralizing antibodies that protect guinea pigs against a severe challenge with the cognate virus (2). In some instances, however, although the total reactivity of antibodies evaluated in immunochemical tests such as enzyme-linked immunosorbent assay is high, the level of neutralizing antibodies is low. This important problem has generated numerous studies aimed at enhancing the immunogenicity of this peptide with the hope of developing a peptide construct that could be used successfully as a synthetic vaccine (3). Recently, we have shown that antisera raised in rabbits against peptides corresponding to the sequence 141-159 of two variants of serotype A cross-reacted strongly with the corresponding retro-inverso analogue peptides. Most importantly, the retro-inverso analogues, also called retro all-D or retro-enantio peptides (4, 5), induced greater and longer-lasting antibody titers than did the respective parent peptides (6). Moreover, we showed with the FP variant analogue (which contains Phe and Pro residues at positions 148 and 153, respectively) that a single inoculation of the retro-inverso peptide elicited high levels of neutralizing antibodies that persisted longer than those induced against the corresponding L-peptide and protected guinea pigs challenged with the cognate virus (7). The enhanced immunogenic reactivities of retro-inverso analogues may result from their higher resistance to proteolysis in biological fluids (7). However, this property does not explain why in immunochemical assays, retro-inverso peptides are often better recognized than the parent peptide by anti-virus, anti-protein, or anti-peptide antibodies with equilibrium affinity cons...

Correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY) and NOE spectroscopy (NOESY) experiments have been used to assign sequentially the 'H 500-MHz NMR spectra of a non-specific (ns) lipid-transfer protein extracted from maize seeds. The spin-system identification and sequential assignment were combined with secondary-structure determination to identify most of the proton resonances of this 93-residue protein. From the sequential connectivities it was established that the secondary structure mainly involved four helical fragments: H1, H2, H3 and H4. This secondary structure was compared with that of wheat ns-lipid-transfer protein recently determined. The four helices are located in nearly the same regions, but helix H4 is appreciably longer in the maize protein than in the wheat protein.Comparison of the transfer activities reveals that the maize protein is more efficient than the wheat nh-lipid-transfer protein and that this difference is probably due to the affinity of the lipid for the binding site and not to the interfacial activation, i.e. adsorption of the ns-lipid-transfer protein to the membrane. From these results, it is suggested that helix H4 is a part of the lipid-binding site or contributes to the folding of this site.The present data define the basis for a further modelling of the three-dimensional structure of the maize ns-lipid-transfer protein which will be compared with that of the wheat ns-lipid-transfer protein in order to establish structure/activity relationships for this class of carriers by using natural ns-lipid-transfer protein mutants.Lipid-transfer proteins form a broad family of proteins, present in micro-organisms, plants and animals. Their main characteristic property is to facilitate inter membrane transfer of lipids in vitro. Some lipid-transfer proteins, such as the phosphatidylinositol-phosphatidylcholine transfer protein from bovine liver, transfer more specifically phospholipids, whereas others are able to transfer phospholipids, glycolipids, fatty acids and sterols (see [l-31 for recent reviews). All the plant lipid-transfer proteins extracted from different sources and organs belong to the family of non-specific (ns) lipid-transfer proteins, since they are able to transfer various phospholipids as well as galactosyldiacylglycerols and they also bind free fatty acids [l].More than I S primary structures of plant ns-lipid-transfer proteins have been determined and a consensus sequence has been recently established [4]. The eight Cys residues present in these sequences are strictly conserved and the positions of the four disulfide bridges has been determined in the castor bean ns-lipid-transfer protein [S]. It can then be anticipated that these covalent links should play a similar role in the Abbreviations. COSY, correlation spectroscopy ; 2QF, double quantum filtered; 24, double quantum ; 3QF, triple quantum filtered; TOCSY, total correlation spectroscopy; NOESY, NOE spectroscopy; ns, non-specific. overall folding of the polypeptide backbones. ns-lipidtrans...