(597c) Effects of Native Disulfide Reduction On Unfolding and Non-Native Aggregation of Alpha-Chymotrypsinogen A

Proper disulfide formation can be essential for the conformational stability of natively folded proteins. Often, the effects of disulfide bonds are studied from the perspective of protein refolding from inclusion bodies or other misfolded states. The present study focuses instead on a comparison of unfolding and aggregation of alpha-chymostrypsinogen A with (aCgn) and without (aCgnSH) one of its native disulfides (C191-C220) intact. The effects of losing this disulfide are shown in terms of the thermodynamics of unfolding, kinetics and mechanism of aggregation, and structure and morphology of the resulting aggregates. In terms of unfolding thermodynamics, aCgnSH has lower conformational stability, in keeping with estimates from other model systems. Aggregate morphology and structure are similar or indistinguishable for aCgn and aCgnSH by far-UV circular dichroism, intrinsic fluorescence and ThT binding, and multi-angle static laser light scattering. In addition, aggregates of aCgn and aCgnSH are both able to cross-seed with monomers of the other species. However, unlike aCgn, aggregates of aCgnSH are highly resistive to dissociation even under extremes of temperature and denaturant concentration ? they ultimately require high hydrostatic pressure to facilitate dissociation. Finally, folded aCgnSH not only is less conformationally stable than aCgn monomer, but also has an altered pathway for aggregate nucleation. Quantitative and qualitative analysis of aggregation kinetics shows that the stoichiometry of the nucleus for creating new aggregates is significantly smaller for aCgnSH than for aCgn. The results highlight the importance of disulfide linkages not only for monomer conformational stability, but also for restricting the conformational space of unfolded monomers as they search for misfolded and aggregation-competent conformers.