Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain

The transition state for the folding pathway of the activation domain of human procarboxypeptidase A2 (ADA2h) has been analyzed by the protein engineering approach. Recombinant ADA2h is an 81-residue globular domain with no disulfide bridges or cis-prolyl bonds, which follows a two-state folding transition. Its native fold is arranged in two alpha-helices packing against a four-stranded beta-sheet. Application of the protein engineering analysis for 20 single-point mutants spread throughout the whole sequence indicates that the transition state for this molecule is quite compact, possessing some secondary structure and a hydrophobic core in the process of being consolidated. The core (folding nucleus) is made by the packing of alpha-helix 2 and the two central beta-strands. The other two strands, at the edges of the beta-sheet, and alpha-helix 1 seem to be completely unfolded. These results, together with previous analysis of ADA2h with either of its two alpha-helices stabilized through improved local interactions, suggest that alpha-helix 1 does not contribute to the folding nucleus, even though it is partially folded in the denatured state under native conditions. On the other hand, alpha-helix 2 folds partly in the transition state and is part of the folding nucleus. It is suggested that a good strategy to improve folding speed in proteins would be to stabilize the helices that are not folded in the denatured state but are partly present in the transition state. Comparison with other proteins shows that there is no clear relationship between fold and/or size with folding speed and level of structure in the transition state of proteins.