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Folding mechanism of an ankyrin repeat protein : scaffold and active site formation of human CDK inhibitor p19(INK4d)

Title data

Löw, Christian ; Weininger, Ulrich ; Zeeb, Markus ; Zhang, Wei ; Laue, Ernest D. ; Schmid, Franz X. ; Balbach, Jochen:
Folding mechanism of an ankyrin repeat protein : scaffold and active site formation of human CDK inhibitor p19(INK4d).
In: Journal of Molecular Biology. Vol. 373 (12 October 2007) Issue 1 . - pp. 219-231.
ISSN 0022-2836
DOI: https://doi.org/10.1016/j.jmb.2007.07.063

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Abstract in another language

The p19(INK4d) protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by inhibiting the cyclin D-dependent kinases (CDK) 4 and 6. We investigated the folding of p19(INK4d) by urea-induced unfolding transitions, kinetic analyses of unfolding and refolding, including double-mixing experiments and a special assay for folding intermediates. Folding is a sequential two-step reaction via a hyperfluorescent on-pathway intermediate. This intermediate is present under all conditions, during unfolding, refolding and at equilibrium. The folding mechanism was confirmed by a quantitative global fit of a consistent set of equilibrium and kinetic data revealing the thermodynamics and intrinsic folding rates of the different states. Surprisingly, the N<-->I transition is much faster compared to the I<-->U transition. The urea-dependence of the intrinsic folding rates causes population of the intermediate at equilibrium close to the transition midpoint. NMR detected hydrogen/deuterium exchange and the analysis of truncated variants showed that the C-terminal repeats ANK3-5 are already folded in the on-pathway intermediate, whereas the N-terminal repeats 1 and 2 are not folded. We suggest that during refolding, repeats ANK3-ANK5 first form the scaffold for the subsequent assembly of repeats ANK1 and ANK2. The binding function of p19(INK4d) resides in the latter repeats. We propose that the graded stability and the facile unfolding of repeats 1 and 2 is a prerequisite for the down-regulation of the inhibitory activity of p19(INK4d) during the cell-cycle.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 17804013
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: Yes
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 23 Apr 2015 09:47
Last Modified: 28 May 2015 15:29
URI: https://eref.uni-bayreuth.de/id/eprint/10626