Thermal, chemical and chemothermal denaturation of yeast enolase

We studied the temperature- and denaturant-induced denaturation of yeast enolase by means of Fourier transform in- frared spectroscopy. The temperature-induced denaturation/aggregation of the enzyme in the absence of denaturant was highly cooperative and occurred between 55 and 65◦C with a midpoint of ∼58◦C. Above 55◦C, the intensity at 1656 cm−1 (pre- dominantly α-helix) decreases as a function of temperature, accompanied by the appearance of two new bands at 1622 and 1696 cm−1, indicating the formation of intermolecular β-sheet aggregates. Five clearly defined isosbestic points were ob- served, indicating a two-state conformational transition. Addition of a non-denaturing concentration of gdnHCl (0.4 M) caused the thermal denaturation/aggregation of the enzyme to proceed faster, but this revealed no unfolding intermediate. The gdnHCl- induced unfolding was first detected at a gdnHCl concentration of above 0.4 M, evidenced by loss of α-helix and β-sheet structures as functions of denaturant concentration. The fully unfolded state was reached at a gdnHCl concentration of 1.6 M. A significant amount of intermolecular β-sheet aggregate was detected at gdnHCl concentrations between 0.6 and 1.0 M, which disappeared as the denaturant concentration increased further. The gdnHCl-unfolded state is a heterogeneous ensemble of turns, helix/loops, and random structures, which continues to change at higher concentrations of denaturant.