Conformational flexibility and folding of synthetic peptides representing an interdomain segment of polypeptide chain in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

Synthetic peptides (32 residues in length) were synthesized with amino acid sequences identical to, or related to, the long (alanine + proline)-rich region of polypeptide chain that links the innermost lipoyl domain to the dihydrolipoamide dehydrogenase-binding domain in the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The 400-MHz 1H NMR spectra of the peptide (Mr approximately 2800) closely resembled the sharp resonances in the spectrum of the intact complex (Mr approximately 5 x 10(6], and the apparent pKa (6.4) of the side chain of a histidine residue in one of the peptides was found to be identical to that previously observed for a histidine residue inserted by site-directed mutagenesis into the corresponding position in the same (alanine + proline)-rich region of a genetically reconstructed enzyme complex. These results strongly support the view that the three long (alanine + proline)-rich regions of the dihydrolipoyl acetyltransferase chains are exposed to solvent and enjoy substantial conformational flexibility in the enzyme complex. More detailed analysis of the peptides by circular dichroism and by 1H and 13C NMR spectroscopy revealed that they were disordered in structure but were not random coils. In particular, all the Ala-Pro peptide bonds were greater than 95% in the trans configuration, consistent with a stiffening of the peptide structure. Differences in the sequences of the three long (alanine + proline)-rich segments may reflect structural tuning of these segments to optimize lipoyl domain movement in enzyme catalysis.