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Impact of four 13C-proline isotope labels on the infrared spectraof ribonuclease T1

Item Type:Article
Title:Impact of four 13C-proline isotope labels on the infrared spectraof ribonuclease T1
Creators Name:Moritz, R. and Fabian, H. and Hahn, U. and Diem, M. and Naumann, D.
Abstract:Ribonuclease T1 was biosynthesized, with all four prolines 13C-labeled in the peptide C=O bond, using a proline auxotrophic yeast strain of Saccharomyces cerevisiae. The 13C- and 12C-proline isotopomers of ribonuclease T1 were investigated by infrared spectroscopy in the thermally unfolded and natively folded state at 80 and 20 °C, respectively. In the thermally unfolded state, both proteins established almost indistinguishable spectral features in the secondary structure sensitive amide I region. In contrast, the spectra measured at 20 °C revealed substantial qualitative and quantitative differences, though parallel analysis by circular dichroism suggested identical native folds for both isotopomers. Major spectral differences in the infrared spectra were detected at 1626 and 1679 cm-1, which are diagnostic marker bands for antiparallel β-sheets in ribonuclease T1 and at 1645 cm-1, a region that is characteristic for the infrared absorption of irregular structures. Starting with the known three-dimensional structure of ribonuclease T1, the observed effects of the isotope labeling are discussed on the basis of transition dipole coupling between the 12C=O and 13C=O groups. The experimental results were confirmed by transition dipole coupling calculations of the amide I manifold of the labeled and unlabeled variant.
Keywords:Aspergillus Oryzae, Carbon Isotopes, Circular Dichroism, Fourier Transform Infrared Spectroscopy, Isotope Labeling, Molecular Models, Proline, Protein Conformation, Ribonuclease T1, Saccharomyces Cerevisiae
Source:Journal of the American Chemical Society
Publisher:American Chemical Society
Page Range:6259-6264
Date:1 June 2002
Official Publication:https://doi.org/10.1021/ja011749s
PubMed:View item in PubMed

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