Thermal unfolding of ribonuclease A in phosphate at neutral pH: deviations from the two-state model.
The thermal denaturation of ribonuclease A (RNase A) in the presence of phosphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existence of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to monitor changes in the secondary structure. Changes in the tertiary structure were monitored by near-uv CD. Spectral bandshape changes with change in temperature were analyzed using factor analysis. The global unfolding curves obtained from DSC confirmed that structural changes occur in the molecule before the main thermal denaturation transition. The analysis of the far-uv CD and FTIR spectra showed that these lower temperature-induced modifications occur in the secondary structure. No pretransition changes in the tertiary structure (near-uv CD) were observed. The initial changes observed in far-uv CD were attributed to the fraying of the helical segments, which would explain the loss of spectral intensity with almost no modification of spectral bandshape. Separate analyses of different regions of the FTIR amide I' band indicate that, in addition to alpha-helix, part of the pretransitional change also occurs in the beta-strands.
Stelea
S D
SD
Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, USA.
Pancoska
P
P
Benight
A S
AS
Keiderling
T A
TA
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013816:Thermodynamics
D012259:Ribonuclease, Pancreatic