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

Abstract

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased ␤-sheet and reduced ␣-helix content relative to the native state. Urea-induced unfolding at 25°C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. 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 little temperature dependence from 15°to 60°C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25°C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Is There or Isn't There? The Case for (and Against) Residual Structure in Chemically Denatured Proteins

“…These regions happen to be the adjacent energy wells in the upper left quadrant of the Ramachandran energy landscape, which typically correspond to the β-sheet and P II helix. Experimental support for the enthalpically favorable nature of the P II well includes the observation that, as urea denatured T7 RNA polymerase is heated, the P II denatured structure will start to move toward a more randomized population of conformations, whereas upon cooling, P II structure is stabilized (Griko et al, 2001). Detailed analysis of this hypothesis via atomistic simulations parallels the experimental results.…”

Recombinant expression of ShPI-1A, a non-specific BPTI-Kunitz-type inhibitor, and its protection effect on proteolytic degradation of recombinant human miniproinsulin expressed in Pichia pastoris

“…We also studied the conformational stability of rShPI-1A using different concentrations of urea as denaturing agent. A full secondary structure analysis was not possible, as the absorption by urea and chloride ions precluded measurements below 200 nm (Griko et al, 2001). The secondary structure was partially affected by the addition of urea (Fig.…”

Residue-level NMR View of the Urea-driven Equilibrium Folding Transition of SUMO-1 (1-97): Native Preferences Do Not Increase Monotonously

“…The local preferences could vary, depending upon the environmental conditions. In view of this, there have been several efforts to characterize the unfolded states in vitro and the intermediates created by different means, which include chemical denaturation, [14][15][16][17][18][19][20][21] pH denaturation, [22][23][24] thermal denaturation, 25,26 etc.…”