Thermodynamic characterization of the human acidic fibroblast growth factor: Evidence for cold denaturation

The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrati ons of urea using fluorescence and far-UV CD circular dichroism (CD) spectr oscopy. The equilibrium unfolding transitions at pH 7.0 are adequately desc ribed by a two-state (native tt unfolded state) mechanism. The stability of the protein is pH-dependent, and the protein unfolds completely below pH 3 .0 Cat 25 degreesC). hFGF-1 is shown to undergo a two-state transition only in a narrow pH range (pH 7.0-8.0). Under acidic (pH <6.0) and basic (pH > 8.0) conditions, hFGF-1 is found to unfold noncooperatively, involving the accumulation of intermediates. The average temperature of maximum stability is determined to be 295.2 K. The heat capacity change (DeltaC(p)) for the unfolding of hFGF-1 is estimated to be 2.1 +/- 0.5 kcal mol(-1) K-1. Temper ature denaturation experiments in the absence and presence of urea show tha t hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional H-1-N -15 HSQC spectra of hFGF-1 acquired at subzero temperatures clearly show th at hFGF-1 unfolds under low-temperature conditions. The significance of the noncooperative unfolding under acidic conditions and the cold denaturation process observed in hFGF-1 are discussed in detail.