Thermodynamics of Protein Self-Association and Unfolding. The Case of Apolipoprotein A-I

Abstract

Protein self-association and protein unfolding are two temperature-dependent processes whose understanding is of utmost importance for the development of biological pharmaceuticals since protein association may stabilize or destabilize protein structure and function. We present new theoretical and experimental methods to analyze the interrelation between self-association and unfolding. We introduce isothermal dilution calorimetry as a new method to quantify protein self-association. Using binding partition functions we present thermodynamic expressions to describe cooperative protein association equilibria. We measured protein unfolding with differential scanning calorimetry and analyzed the unfolding process in terms of the cooperative Zimm-Bragg theory which appears to be well suited for α-helical proteins. As a model protein we chose human recombinant apolipoprotein A-I. Apo A-I self-association and unfolding were investigated with isothermal titration calorimetry, differential scanning calorimetry, analytical ultracentrifugation, and circular dichroism spectroscopy. A consistent quantitative interpretation of all experimental results could be given in terms of cooperative self-association and unfolding. Self-association had a maximum at 21°C with an association constant Ka = 5.6 x 105 M−1. Unfolding occurred between 45°C and 65°C and was reversible and independent of protein concentration up to 160 x 10−6 M. The midpoint of unfolding was at 52-53°C and the enthalpy of thermal unfolding was 420 kJ/mol. The molar heat capacity increased by 5.1 kJ/molK upon unfolding corresponding to a loss of 80 to 85 helical segments, as was confirmed by CD spectroscopy. The temperature dependence of the interrelated association and unfolding equilibria was dominated by the large enthalpy of unfolding. Thermodynamic analysis predicted and experimental results confirmed that Apo A-I unfolding and Apo A-I oligomer dissociation occurred simultaneously.