Conformational analysis of peptide fragments derived from the peripheral subunit binding domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: Evidence for nonrandom structure in the unfolded state

There is currently a great deal of interest in the early events in protein folding. Two issues that have generated particular interest ape the nature of the unfolded state under native conditions and the role of local interac tions in folding. Here, we report the results of a study of a set of peptid es derived from a small two-helix protein, the peripheral subunit-binding d omain of the pyruvate dehydrogenase multienzyme complex. Five peptides of o verlapping sequence were prepared, including sequences corresponding to eac h of the helices and to the region connecting them. The peptides were chara cterized by CD and, where possible, nmr. A peptide corresponding to the sec ond helix is between 12 and 17% helical at neutral pH. CD also indicates a lower percentage of helical structure in the peptide corresponding to the f irst cu-helix, although the values of the ct-proton chemical shifts suggest some preference for nonrandom structure. Peptides corresponding to the int erhelical loop, which in the full domain contains two overlapping beta-turn s and a 5-residue 3(10)-helix, are less structured. There is no significant change in the helicity of any of these peptides with pH. To test for fragm ent complementation, CD spectra of the two peptides derived from each helix and the long connecting peptide were compared to the spectra of each possi ble pair, as well as to a mixture containing all three. No increase in stru cture was observed. We complement our peptide studies by characterizing a p oint mutant, D34V, which disrupts a critical hydrogen bonding network. This mutant is unable to fold and provides a useful model of the denatured stat e. The mutant is between 9 and 16 % helical as judged by CD. The modest amo unt of helical structure formed in some of the peptide fragments and in the point mutant suggests that the denatured state of the peripheral subunit b inding domain is not completely unstructured. This may contribute to the ve ry rapid folding observed for the intact protein. (C) 1999 John Wiley Br So ns, Inc.