Thermodynamic Stability of Annexin V E17G:  Equilibrium Parameters from an Irreversible Unfolding Reaction

Conformational stability of the membrane-binding protein annexin V E17G has been determined by high-sensitivity differential scanning microcalorimetry (DSC) measurements and by isothermal, guanidinium hydrochloride (GdnHCl)-induced unfolding studies. Wild-type annexin V and the E17G mutant protein studied here are structurally almost identical. Therefore, it can be expected that the present results will not deviate significantly from the stability data of the wild-type molecule. Thermal unfolding is irreversible, while GdnHCl unfolding shows a high degree of reversibility. We were able to demonstrate that characteristic features of annexin V E17G unfolding permit us to extract from the kinetically controlled heat capacity curves thermodynamic equilibrium parameters at the high heating rates. The thermodynamic quantities obtained from the DSC studies in phosphate buffer at pH 7.0 are as follows: t 1/2 = 54.7 °C (heating rate of 2.34 K min-1), ΔH° = 690 kJ mol-1, and ΔC p = 10.3 kJ mol-1 K-1 which correspondends to a value of ΔG°D (20 °C) of 53.4 kJ mol-1. When compared on a per gram basis, these thermodynamic parameters classify annexin V E17G as a marginally stable protein. This conclusion is consistent with structural and functional features of the protein that require conformational adaptability for hinge-bending motions and pore formation on interaction with membranes. We observed a large difference between the change in the Gibbs energy value derived from the heat capacity studies and that determined from the GdnHCl unfolding curve. The difference appears to stem from a specific interaction of the protein with the denaturant that results in both a low half-denaturation concentration c 1/2 of 1.74 M and a small slope (6.0 kJ L mol-2) of the ΔG app versus [GdnHCl] plot. The extraordinary interaction of annexin V with GdnHCl is also manifested in the enormous depression of the transition temperature Δt 1/2 (=18 °C) when the GdnHCl concentration is increased from 0 to 1 M. “Regular” proteins experience an average decrease in the transition temperature of 8 ± 2 °C per 1 M change in the concentration of GdnHCl.