Affiliations: Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, CO 80639, USA
Note: [] Present address: Atrix Laboratories, Inc., 2579 Midpoint Drive, Fort Collins, CO 80522, USA.
Note: [] Corresponding author: Department of Chemistry and Biochemistry, University of Northern Colorado, Greeley, CO 80639, USA. Tel.: +1 970 351 1284; Fax: +1 970 351 1269; E‐mail: adong@unco.edu.
Abstract: We studied the temperature‐ and denaturant‐induced denaturation of yeast enolase by means of Fourier transform infrared 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 (predominantly 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 observed, 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.
Journal: Spectroscopy, vol. 17, no. 2-3, pp. 453-467, 2003
