Nativelike structure and stability in a truncation mutant of a protein minidomain: The peripheral subunit-binding domain

Despite its small size, the peripheral subunit-binding domain from the dihy drolipoamide acetyltransferase component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex adopts a unique, compact struct ure. To determine whether the full 33 residue sequence is required for the domain to adopt a stable, nativelike structure, 3 proteins of different len gths were prepared. Psbd41 corresponds to residues 3-43 of the domain, psbd 36 spans residues 6-41, and psbd33 comprises residues 7-39, Psbd41 folds in a cooperative, two-state fashion with a T-m of 53 degrees C and a stabilit y at 25 degrees C of 2.2 kcal mol(-1). Psbd36 is nearly as stable with a T- m of 48 degrees C and a stability of 1.8 kcal mol(-1). Similar m-values and heat capacities suggest that psbd36 and psbd41 bury approximately the same surface area. Minimal differences in CalphaH and NH chemical shifts betwee n psbd41 and psbd36 show that the two sequences adopt the same tertiary fol d. On a per residue basis, Delta H degrees and Delta C(p)degrees fall withi n the range typical for single-domain globular proteins, Psbd33 is signific antly less stable. It is not fully folded at 25 degrees C, and at all tempe ratures it shows broadened NMR lines. ANS titrations provide evidence that this is due to an equilibrium between nativelike and unfolded molecules rat her than formation of a molten globule. The fraction of psbd33 molecules wh ich are folded appear to adopt the same structure as the full-length domain . Thus, although more than the 33 residue core is required to form a fully stable native structure, the entire sequence is not required for folding.