A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins.

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-Arg-Tyr-NH, was designed and applied to the detection of folding intermediates of model proteins.

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism.

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 Lys residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase.

The propeptide sequence assists the correct folding required for the enzymatic activity of cocoonase.

Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail.