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 acetyl transferase (E2) component of the pyruvate dehydrogenase multienzyme c omplex from Bacillus stearothermophilus is stably folded, despite its short sequence of only 43 amino acid residues. A 41 residue peptide de rived from this domain, psbd41, undergoes a cooperative thermal unfold ing transition with a t(m) of 54 degrees C. This three-helix protein i s monomeric as judged by ultracentrifugation and concentration-depende nt CD measurements. Peptides corresponding to the individual helices a re largely unstructured both alone and in combination, indicating that the unusual stability of this protein does not arise solely from unus ually stable alpha-helices. Chemical denaturation by guanidine hydroch loride 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 resid ue at position 34 that may provide stability and specificity to the fo ld. A mutant peptide, psbd41Asn was synthesized in which the buried as partate residue was mutated to asparagine. This peptide still folds co operatively and it is monomeric, but is much less thermostable than th e wild-type with a t(m) of only 31 degrees C. Chemical denaturations a t 4 degrees C give an m-value of 740 cal mol(-1) M-1, similar to the w ild-type, but the stability Delta G(H2O) is only 1.4 kcal mol(-1). Bot h the wild-type and the mutant unfold at extremes of pH, but at 4 degr ees 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 t he wild-type and it binds ANS. These results show that Asp34 is vital for the stability and specificity of this structure, the second smalle st natural sequence known to ford in the absence of disulfide bonds or metal or ligand-binding sites. (C) 1998 Academic Press Limited.