Ribonuclease A revisited: infrared spectroscopic evidence for lack of native-like secondary structures in the thermally denatured state.
To address a number of conflicting reports in the literature, we undertook an infrared spectroscopic study to test for the presence of native-like secondary structures in thermally denatured ribonuclease A. Ribonuclease A does not aggregate at high temperatures, and the infrared spectrum shows a completely featureless amide I band contour. Using 13C-labeled urea, we were also able to obtain the infrared spectrum of the chemically denatured protein, which is practically identical with that of the heat-denatured protein. To the best of our knowledge, this is the first study that uses 13C-labeled urea as a chemical denaturant which circumvents the problem encountered with the strong absorption of urea in the conformation-sensitive amide I region of proteins; it opens up the possibility of investigating protein folding/unfolding processes in the presence of high concentrations of chemical denaturants. From an analysis of the amide I region of the infrared spectra of thermally and chemically denatured RNase A, it was concluded that heat-denatured ribonuclease A does not contain any significant amount of authentic hydrogen-bonded secondary structures. Furthermore, a comparison of the infrared spectra of ribonuclease A with those of ribonuclease T1 demonstrates that in spite of major differences between their native structures there are practically no differences between their heat-denatured states. This would not be expected if there were residual native-like secondary structures in the thermally denatured state of one or both of these proteins.
Fabian
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Institute for Biochemistry, Humboldt-University Berlin, Max-Delbrück-Center for Molecular Medicine, Germany.
Mantsch
H H
HH
D002247:Carbon Isotopes; D006358:Hot Temperature; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D006163:Ribonuclease T1 / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013055:Spectrophotometry, Infrared; D013816:Thermodynamics; D014508:Urea
D002247:Carbon Isotopes; D014508:Urea; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic