Premium
Thermal unfolding of ribonuclease A in phosphate at neutral pH: Deviations from the two–state model
Author(s) -
Stelea Simona D.,
Pancoska Petr,
Benight Albert S.,
Keiderling Timothy A.
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.47101
Subject(s) - chemistry , circular dichroism , protein secondary structure , fourier transform infrared spectroscopy , differential scanning calorimetry , ribonuclease , analytical chemistry (journal) , denaturation (fissile materials) , crystallography , amide , rnase p , nuclear chemistry , chromatography , organic chemistry , thermodynamics , optics , biochemistry , rna , physics , gene
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 α‐helix, part of the pretransitional change also occurs in the β‐strands.