Marginal stability drives irreversible unfolding of large multi-domain family 3 glycosylhydrolases from thermo-tolerant yeast.

Protein folding is an extremely complex and fast, yet perfectly defined process, involving interplay of many intra and inter-molecular forces. In vitro, these molecular interactions are reversible for many proteins e.g., smaller and monomeric, organized into single domains. However, refolding of larger multi-domain/multimeric proteins is much more complicated, proceeds in a hierarchal way and is often irreversible. In a comparative study on two large, multi-domain and multimeric isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from Pichia etchellsii, we studied spontaneous and assisted refolding under three denaturing conditions viz. GdnHCl, alkaline pH and heat. During refolding, higher refolding yields were obtained for BGLII in case of pH induced unfolding (13.89%±0.25) than BGLI (6%±0.85) while for GdnHCl induced unfolding, refolding was marginal (BGLI=5%±0.5; BGLII=6%±0.69). Thermal unfolding was irreversible while assisted refolding also showed little structural gain for both proteins. When the apparent free energies of unfolding (ΔG) were calculated from GdnHCl unfolding data, their values were strikingly found to be lower (BGLI ΔG=3.02kcal/mol; BGLII ΔG=2.99kcal/mol) than reported for globular (ΔG=5-15kcal/mol)/multimeric proteins (ΔG=23-29kcal/mol) indicating marginal stability results in low refolding.