Two peptides (PEP1, 26 residues, and PEP2, 22 residues) were synthesized with amino acid sequences identical to two of the long segments of polypeptide chain rich in alanine, proline, and charged amino acids that link the lipoyl domains together in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The circular dichroism and 400-MHz 1H NMR spectra of the peptides indicated that they lacked regular secondary structure. Even in the presence of 45% (v/v) hexafluoroisopropanol, they appeared to acquire a helical content of only 23-25%. However, 13C NMR spectroscopy revealed that the Ala-Pro peptide bonds were all (> 95%) in the trans configuration, compared with a value of 87% for the Ala-Pro bond in the model peptide AAPA, which is a recurrent sequence motif in PEP1 and PEP2. Likewise in peptides representing the N- and C-terminal halves of peptide PEP2, the Ala-Pro bonds were again all (> 95%)-trans, suggesting that peptide length is the essential determinant of the cis:trans ratio. Antisera were raised against peptides PEP2 and PEP3, the latter representing a third interdomain segment of polypeptide chain (Radford, S. E., Laue, E. D., Perham, R. N., Martin, S. R., and Appella, E. (1989a) J. Biol. Chem. 264, 767-775). Despite extensive sequence similarity among peptides PEP1, PEP2, and PEP3, only limited immunological cross-reactivity was observed, which suggests that the antigenic epitope(s) in the peptides are different and distinct. It is likely that these peptides are representative of a class of inter-domain linkers or spacers found in a wide variety of proteins and endowed with varying degrees of flexibility and stiffness to match their particular biological purpose.