Thermal unfolding of ribonuclease A in phosphate at neutral pH: Deviationsfrom the two-state model

Citation
Sd. Stelea et al., Thermal unfolding of ribonuclease A in phosphate at neutral pH: Deviationsfrom the two-state model, PROTEIN SCI, 10(5), 2001, pp. 970-978
Citations number
50
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN SCIENCE
ISSN journal
0961-8368 → ACNP
Volume
10
Issue
5
Year of publication
2001
Pages
970 - 978
Database
ISI
SICI code
0961-8368(200105)10:5<970:TUORAI>2.0.ZU;2-0
Abstract
The thermal denaturation of ribonuclease A (RNase A) in the presence of pho sphate at neutral pH was studied by differential scanning calorimetry (DSC) and a combination of optical spectroscopic techniques to probe the existen ce of intermediate states. Fourier transform infrared (FTIR) spectra of the amide I' band and far-uv circular dichroism (CD) spectra were used to moni tor changes in the secondary structure. Changes in the tertiary structure w ere monitored by near-uv CD. Spectral bandshape changes with change in temp erature were analyzed using factor analysis. The global unfolding curves ob tained from DSC confirmed that structural changes occur in the molecule bef ore 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 e xplain the loss of spectral intensity with almost no modification of spectr al bandshape. Separate analyses of different regions of the FTIR amide I' b and indicate that, in addition to alpha -helix, part of the pretransitional change also occurs in the beta -strands.