An important event in the generation of an immune response is the activation of T cells by peptides bound to the class II proteins of the major histocompatiblity complex (MHC). Binding of a peptide to an MHC protein is stabilized by multiple interactions between the protein, peptide side-chains, and peptide backbone. Unstable protein-peptide complexes that precede formation of long-lived complexes are presumably engaged in a smaller number of these binding interactions. To investigate the effect of peptide structure on the formation of unstable complexes, we have strategically modified a peptide that forms only long-lived complexes (dissociation rate constant k(off) = 2.5 x 10(-6) s(-1)). Dissociation of the modified peptide from an MHC protein is biphasic with dissociation rate constants k(off) = 5.3 x 10(-4) and 2.6 x 10(-6) s(-1). Thus, at least two detectable complexes are formed. These results demonstrate that changes in peptide structure alone are sufficient to result in the formation of isomeric structures of MHC protein-peptide complexes.