Cooperative folding of a protein mini domain: The peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex

The peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide derived from this domain, psbd41, undergoes a cooperative thermal unfolding transition with a t(m) of 54 degrees C. This three-helix protein is monomeric as judged by ultracentrifugation and concentration-dependent CD measurements. Peptides corresponding to the individual helices are largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unusually stable alpha-helices. Chemical denaturation by guanidine hydrochloride is also cooperative with a Delta G(H2O) of 3.1 kcal mol(-1) at pH 8.0 and 25 degrees C. The chemical denaturation is broad with an m-value of 760 cal mol(-1) M-1. psbd41 contains a buried aspartate residue at position 34 that may provide stability and specificity to the fold. A mutant peptide, psbd41Asn was synthesized in which the buried aspartate residue was mutated to asparagine. This peptide still folds cooperatively and it is monomeric, but is much less thermostable than the wild-type with a t(m) of only 31 degrees C. Chemical denaturations at 4 degrees C give an m-value of 740 cal mol(-1) M-1, similar to the wild-type, but the stability Delta G(H2O) is only 1.4 kcal mol(-1). Both the wild-type and the mutant unfold at extremes of pH, but at 4 degrees C psbd41Asn is folded over a narrower pH range than the wild-type. Although the mutant unfolds cooperatively by thermal and by chemical denaturation, its NMR spectrum is significantly broader than that of the wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smallest natural sequence known to ford in the absence of disulfide bonds or metal or ligand-binding sites. (C) 1998 Academic Press Limited.