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A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy.

Title data

Balbach, Jochen ; Steegborn, Clemens ; Schindler, Thomas ; Schmid, Franz X.:
A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy.
In: Journal of Molecular Biology. Vol. 285 (15 January 1999) Issue 2 . - pp. 829-842.
ISSN 0022-2836
DOI: https://doi.org/10.1006/jmbi.1998.2364

Abstract in another language

The rate-limiting step during the refolding of S54G/P55N ribonuclease T1 is determined by the slow trans-->cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degreesC. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the alpha-helix, beta-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the alpha-helix, and the loops between beta-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N ribonuclease T1 and for cis/trans isomerizations are discussed.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 9878447
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Clemens Steegborn
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 13 Apr 2015 06:13
Last Modified: 13 Apr 2015 06:13
URI: https://eref.uni-bayreuth.de/id/eprint/10000