Most continuous antigenic determinants of tobacco mosaic virus protein (TMVP), myoglobin and lysozyme correspond to those surface regions in the protein structure, as determined by X-ray crystallography, which possess a run of high-temperature factors along the polypeptide backbone, that is, a high segmental mobility. The mobility of an antigenic determinant may make it easier to adjust to a pre-existing antibody site not fashioned to fit the exact geometry of a protein. The correlation found between temperature factors and antigenicity is better than that between hydrophilicity and antigenicity.
In the present study, we developed new turn scales based on the occurrence of amino acids at each of the four positions of a turn using a structural database comprised of 87 proteins. We found that the scales correctly predicted a fraction of the turn regions in proteins with approximately 80% confidence. We used the turn scales for predicting the location of antigenic sites in proteins. The method was developed with the specific aim of predicting only a few peaks for each protein (two or three). We found that it leads to a high level of accurate prediction (70% of correct prediction of known epitopes). Our method should be useful for selecting protein regions to be synthesized in order to produce anti-peptide antibodies cross-reacting with the parent protein.
Three analogues of the model peptide of sequence IRGERA corresponding to the COOH-terminal residues 130-135 of histone H3 were synthesized, and their antigenicity, immunogenicity, and resistance to trypsin were compared to those of the natural L-peptide. The three analogues correspond to the D-enantiomer, containing only D-residues, and two retro-peptides containing NH-CO bonds instead of natural peptide bonds. The chirality of each residue was maintained in the retro-peptide and inverted in the retroinverso-peptide. Antibodies to the four peptide analogues were produced by injecting BALB/c mice with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A. Each of the four peptide analogues induced IgG antibodies of various subclasses. The IgG3 antibodies reacted similarly with the four analogues, whereas antibodies of the IgGl, IgG2a, and IgG2b isotypes showed strong conformational preferences for certain peptides. The retro-inversopeptide IRGERA mimicked the structure and antigenic activity of the natural L-peptide but not of the D-and retro-peptides, whereas the retro-peptide IRGERA mimicked the D-peptide but not the L-and retro-inverso-peptides. The equilibrium affnity constants (Ka) of three monoclonal antibodies generated against the L-and D-peptides with respect to the four peptide analogues were measured in a biosensor system. Large differences in Ka values were observed when each monoclonal antibody was tested with respect to the four peptides. The use of retro-inverso-peptides to replace natural L-peptides is likely to find many applications in immunodiagnosis and as potential synthetic vaccines.The development of neuropeptides, peptide hormones, peptide antibiotics, or peptide-based synthetic vaccines is strongly impaired by the high susceptibility of peptides to proteolysis, which limits, inter alia, parental and oral administration. For many years intense work has been focused on the synthesis of peptide analogues in the search for mimics with enhanced activity and biological half-lives. Examples of modifications introduced in peptides are the replacement of L-amino acid residues by D-amino acids or by unnatural residues (e.g., sarcosine and 3-alanine) and the modification of peptide bonds (1-3). These changes provide pseudopeptides or peptidomimetics with a higher metabolic stability, since most natural proteases cannot cleave D-amino acid residues and nonpeptide bonds. An important problem encountered with such peptide analogues is the conservation of their biological activity. Recently, the D-form of human immunodeficiency virus type 1 protease has been synthesized (4). As could be expected, the enantiomeric protein displayed reciprocal chiral specificity as the enzyme was unable to cleave the normal L-substrate but did hydrolyze its D-enantiomer. In contrast, Wen and Laursen (5) showed that both the L-and D-form of an a-helical antifreeze polypeptide bound equally well to the same achiral ice substrate, whereas Wade et al. (6) found that the L-and D-e...
Differences in the affinity of a monoclonal antibody raised against the protein of tobacco mosaic virus for 15 related peptides (residues 134-146) carrying single-residue modifications were investigated using a novel biosensor technology (Pharmacia BIAcore). Analysis of the peptide-antibody interaction in real time allowed fast and reproducible measurements of both association and dissociation rate constants. Out of 15 mutant peptides analyzed, five were not recognized by the antibody at all, and seven were recognized as well as the wild-type peptide. For three of the peptides, the rate constants were different for the mutant and wild-type peptides. The pattern of residue recognition suggests that the epitope is formed by three residues (140, 143, and 144) in a helical conformation that mimics the structure in the protein. Even a minor modification of these residues totally abolishes recognition by the antibody. Modifications of adjacent residues result in small but significant differences in association and/or dissociation rate constants. One of the recognized residues is totally buried in the three-dimensional structure of TMV protein, suggesting that a structural rearrangement next to the helix occurs during protein-antibody interaction.
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