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 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 beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees 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 degrees to 60 degrees 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 degrees 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.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
Y
Y
Griko
N
N
Sreerama
P
P
Osumi-Davis
R W
RW
Woody
A Y
AY
Woody
D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014508:Urea; D014764:Viral Proteins
D014764:Viral Proteins; D014508:Urea; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases