The use of hydrophobic, alpha-helix-defined peptides in delineating the T cell determinant for pigeon cytochrome c

Abstract

The B10.A T cell proliferative response to pigeon cytochrome c is largely directed to a single site in the molecule located at the carboxyl terminus within the amino acid sequence of residues 81 to 104. This study uses the pigeon cytochrome c-specific T cell clone A.E7 and synthetic peptide analogs to clarify the role of certain residues within this sequence in T cell recognition. By using the helically constrained amino acid, alpha-aminoisobutyric acid, alternated with alanine in an amino-terminal leader sequence, we generated a series of molecules of similar length and alpha-helical conformation but which contain increasing lengths of the native sequence. By comparing the stimulatory ability of this series of peptides, we have clearly identified that the isoleucyl residue at position 95 in pigeon cytochrome c is essential for T cell recognition. This series, when compared with a series containing the same native sequences but without the leader sequence, also showed that the presence of the leader sequence has a general effect on enhancement of T cell recognition. An analysis of the conformational preferences of the peptides using circular dichroism indicated that all of the peptides with leader sequences have a strong preference for the alpha-helical conformation in nonpolar solvents. However, the introduction of helix-breaking residues into these peptides, with a concomitant measured reduction in alpha-helix, did not affect their recognition by clone A.E7. This implies that factors other than conformational stabilization are responsible for the full potency of these peptides. Binding studies to phospholipid vesicles indicated that residues in the leader sequence and in the amino terminus of segment 81-104 beyond residue 95 were important in increasing the ability of the antigens to bind to membranes. These results suggest that the capacity to bind to membranes may be a significant factor in the dose response of T cells to exogenously presented peptides.