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Use of a trypsin-pulse method to study the refolding pathway of ribonuclease

Title data

Lang, Kurt ; Schmid, Franz X.:
Use of a trypsin-pulse method to study the refolding pathway of ribonuclease.
In: European Journal of Biochemistry. Vol. 159 (1 September 1986) Issue 2 . - pp. 275-81.
ISSN 1432-1033
DOI: https://doi.org/10.1111/j.1432-1033.1986.tb09864.x

Official URL: Volltext

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Abstract in another language

Trypsin pulses, applied after varying times of refolding, have been employed to probe the accessibility of the polypeptide chain of ribonuclease A during the process of refolding. The increase in resistance against proteolytic cleavage was measured by activity assays and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The sites of cleavage which become inaccessible in the course of refolding are located in the 31 - 39 chain segment of the ribonuclease chain. Protection of this region against attack by trypsin is attained on the major slow refolding pathway in parallel with the formation of a native-like folded, active intermediate, when refolding is carried out under conditions which strongly stabilize the folded state. Under conditions of marginal stability intermediates are not observed during refolding and the formation of trypsin-resistant molecules occurs with the same kinetics as the generation of native ribonuclease. In the native protein the 31 - 39 region of the ribonuclease chain largely forms a loop structure, which is located at the surface of the molecule. Our results indicate that this part of the sequence is still accessible at early stages of refolding, when a hydrogen-bonded network is formed. It is structured and hence does not become inaccessible until the formation of the overall folded native or native-like structure. This suggests that the 31 - 39 region of the ribonuclease chain is not important for early steps which direct the pathway of refolding.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 3758063
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors > Professorship Biochemistry - Univ.-Prof. Dr. Franz Xaver Schmid
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 > Professorship Biochemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Former Professors
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 30 Apr 2015 12:13
Last Modified: 30 Apr 2015 12:13
URI: https://eref.uni-bayreuth.de/id/eprint/11230