Thermal and urea-induced unfolding in T7 RNA polymerase: Calorimetry, circular dichroism and fluorescence study

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain informati on about the structural organization and the stability of the enzyme. T7RNA P is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative un its that may be regarded as structural domains. interactions between these structural domains and their stability strongly depend on solvent condition s. The unfolding of T7RNAP under different solvent conditions induces a hig hly stable intermediate state that lacks specific tertiary interactions, co ntains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroi sm (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta -sheet and reduced alpha -helix content relative to the native state. Urea-induced unfolding at 25 degreesC reveals a two-step process. The first transition centered near 3 M urea leads to a plateau fr om 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M u rea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows litt le temperature dependence from 15 degrees to 60 degreesC. The second transi tion leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative beca use of conversion of poly(Pro)II to the unordered conformation. Near-ultrav iolet CD spectra at 25 degreesC at varying concentrations of urea are consi stent with this picture. Both thermal and urea denaturation are irreversibl e, presumably because of processes that follow unfolding.