Thermal denaturation profiles of wild-type and mutant EstE1. (A) Far-UV CD spectra of wild-type EstE1 (●), EstE1F276A (■), EstE1F276E (□), EstE1V274A (▲), EstE1V274A/F276A (▯), EstE1L299D (▼), EstE1R270A (▯), EstE1E295A (▯), and EstE1R270A/E295A (▯) are plotted as molar ellipticity versus wavelength. (B) Thermal denaturation profiles of wild-type and mutant EstE1. Changes in molar ellipticity at 222 nm at a scan rate of 1°C/min in the temperature range of 70 to 110°C were measured, and the fractions folded are plotted. (C) Apparent transition temperatures of wild-type and mutant EstE1. Tapp values were estimated by fitting the data shown in (B), using the five-parameter sigmoid function from the curve-fitting program SIGMAPLOT. The inflection point was determined by numerical differentiation of the curves as described previously .