Thermodynamics of denaturation of hisactophilin, a beta-trefoil protein

Hisactophilin is a histidine-rich pH-dependent actin-binding protein from D ictyostelium discoideum. The structure of hisactophilin is typical of the b eta -trefoil fold, a common structure adopted by diverse proteins with unre lated primary sequences and functions. The thermodynamics of denaturation o f hisactophilin have been measured using fluorescence- and CD-monitored equ ilibrium urea denaturation curves, pH-denaturation, and thermal denaturatio n curves, as well as differential scanning calorimetry. Urea denaturation i s reversible from pH 5.7 to pH 9.7; however, thermal denaturation is highly reversible only below pH similar to 6.2. Reversible denaturation by urea a nd heat is well fit using a two-state transition between the native and the denatured states. Urea denaturation curves are best fit using a quadratic dependence of the Gibbs free energy of unfolding upon urea concentration. H isactophilin has moderate, roughly constant stability from pH 7.7 to pH 9.7 ; however, below pH 7.7, stability decreases markedly, most likely due to p rotonation of histidine residues. Enthalpic effects of histidine ionization upon unfolding also appear to be involved in the occurrence of cold unfold ing of hisactophilin under relatively mild solution conditions. The stabili ty data for hisactophilin are compared with data on hisactophilin function, and with data for two other beta -trefoil proteins, human interleukin-1 be ta, and basic fibroblast growth factor.