Thermal unfolding of apolipoprotein A-1. Evaluation of methods and models.

Human apolipoprotein A-1 (Apo A-1) was used as a model protein to compare experimental methods and theoretical models for protein unfolding. Thermal unfolding was investigated in aqueous buffer, in β-octylglucoside solution, and with phospholipid bilayer vesicles. The α-helix content of Apo A-1 increased from 50% in aqueous buffer to 75% in the presence of lipid vesicles, but remained constant in solutions of β-octyl glucoside. Differential scanning calorimetry (DSC) measured the thermodynamic properties of the unfolding process and was our reference method. The increased heat capacity of the unfolded protein made an important contribution to the total enthalpy of unfolding. The structural properties of Apo A-1 were studied with circular dichroism (CD) spectroscopy. The CD-recorded unfolding transitions were broader than the corresponding DSC transitions and were shifted toward higher temperatures. DSC and CD data were analyzed with the two-state model and the Zimm-Bragg theory. The two-state model assumes just two species in solution, native (N) and unfolded (U) Apo A-1. However, Apo A-1 unfolding is a highly cooperative event with helical amino acid residues unfolding and refolding rapidly. For such a sequential process, the Zimm-Bragg theory provides an alternative and physically more realistic model. The Zimm-Bragg theory allowed perfect simulations of the DSC and CD experiments. In contrast, incorrect thermodynamic results were obtained with the two-state model. The Zimm-Bragg theory also provided a physically well-defined analysis of the cooperativity of the folding ⇄ unfolding equilibrium. The cooperative unfolding of Apo A-1 increased upon addition of lipids and decreased in detergent solution.