Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein.

The present work reports a new strategy for triblock-copolymer-assisted refolding of sodium dodecyl sulfate (SDS)-induced unfolded serum protein human serum albumin (HSA) by mixed-micelle formation of SDS with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer EO20PO68EO20 (P123) under physiological conditions. The steady-state and time-resolve fluorescence results show that the unfolding of HSA induced by SDS occurs in a stepwise manner through three different phases of binding of SDS, which is followed by a saturation of interaction. Interestingly, the addition of polymeric surfactant P123 to the unfolded protein results in the recovery of ∼87% of its α-helical structure, which was lost during SDS-induced unfolding. This is further corroborated by the return of the steady-state and time-resolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial nativelike condition. The isothermal titration calorimetry (ITC) data also substantiates that there is almost no interaction between P123 and the native state of the protein. However, the mixed-micelle formation, accompanied by substantial binding affinities, removes the bound SDS molecules from the scaffolds of the unfolded state of the protein. On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refolding the protein back to its nativelike state.