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Thermal unfolding of ribonuclease A in phosphate at neutral pH: Deviations from the two–state model

Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60607-7061, USA

Reprint requests to: Timothy A. Keiderling, Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, Chicago, Illinois 60607-7061; e-mail: tak{at}uic.edu; fax: (312) 996-0431.

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 -helix, part of the pretransitional change also occurs in the ß-strands.

Keywords: Ribonuclease A; protein thermal unfolding; circular dichroism; infrared; differential scanning calorimetry; bandshape analysis

Abbreviations: RNase A, ribonuclease A • DSC, differential scanning calorimetry • FTIR, Fourier transform infrared • CD, circular dichroism • NMR, nuclear magnetic resonance • S/N, signal-to-noise ratio • PC/FA, the principal component method of factor analysis

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