Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

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Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.