Ribonuclease A revisited : infrared spectroscopic evidence for lack of native-like secondary structures in the thermally denatured state

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DOIResolve DOI: http://doi.org/10.1021/bi00041a046
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TypeArticle
Journal titleBiochemistry
ISSN0006-2960
1520-4995
Volume34
Issue41
Pages1365113655; # of pages: 5
SubjectOM; carbon isotopes; hot temperature; protein denaturation; protein folding; protein structure, secondary; ribonuclease T1/chemistry; ribonuclease, pancreatic/chemistry; spectrophotometry, infrared; thermodynamics; urea
AbstractTo 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.
Publication date
PublisherAmerican Chemical Society
LanguageEnglish
AffiliationNational Research Council Canada; NRC Institute for Biodiagnostics
Peer reviewedYes
NRC number291
NPARC number9742705
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Record identifierf051a510-5625-4a02-9ff8-faa3bba77c8a
Record created2009-07-17
Record modified2016-11-21
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