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  Protein folding and stability of human CDK inhibitor p19(INK4d)

Zeeb, M., Rosner, H., Zeslawski, W., Canet, D., Holak, T. A., & Balbach, J. (2002). Protein folding and stability of human CDK inhibitor p19(INK4d). Journal of Molecular Biology, 315(3), 447-457.

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Genre: Journal Article
Alternative Title : J. Mol. Biol.

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 Creators:
Zeeb, M., Author
Rosner, H., Author
Zeslawski, W., Author
Canet, D., Author
Holak, T. A.1, Author              
Balbach, J., Author
Affiliations:
1Holak, Tad / NMR Spectroscopy, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565154              

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Free keywords: p19(INK4d); protein folding intermediate; protein folding; NMR; phenylalanine fluorescence
 Abstract: P19(INK4d) is a tumor suppressing protein and belongs to a family of cyclin D-dependent kinase inhibitors of CDK4 and CDK6, which play a key role in human cell cycle control. P19 comprises ten alpha-helices arranged sequentially in five ankyrin repeats forming an elongated structure. This rather simple topology, combined with its physiological function, makes p19 an interesting model protein for folding studies. Urea-induced unfolding transitions monitored by far-UV CD and phenylalanine fluorescence coincide and suggest a two-state mechanism for equilibrium unfolding. Unfolding of p19 followed by 2D H-1-N-15 HSQC spectra revealed a third species at moderate urea concentrations with a maximum population of about 30% near 3.2 M urea. It shows poor chemical shift dispersion, but cross-peaks emerge for some residues that are distinct from the native or unfolded state. This equilibrium intermediate either arises only at high protein concentrations (as in the NMR experiment) or has similar optical properties to the unfolded state. Stopped-flow far-UV CD experiments at various urea concentrations revealed that alpha-helical structure is formed in three phases, of which only the fastest phase (10 s(- 1)) depends upon the urea concentration. The kinetic of the slowest phase (0.017 s(-1)) can be resolved by 1D real-time NMR and accelerated by cyclophilin. It is limited in rate by prolyl isomerization, and native-like ordered structure cannot form prior to this isomerization. The two fast phases lead to 83% native protein within the dead time of the NMR experiment. In contrast to p16(INK4a), which exhibits only a marginal stability and high unfolding rates, p19 shows the expected stability for a protein of this size with a clear kinetic barrier between the unfolded and folded state. Therefore, p19 might complement the function of less stable INK4 inhibitors in cell cycle control under unfavorable conditions. (C) 2002 Academic Press.

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Language(s): eng - English
 Dates: 2002-01-18
 Publication Status: Published in print
 Pages: -
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: eDoc: 35345
ISI: 000173577400015
 Degree: -

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Title: Journal of Molecular Biology
  Alternative Title : J. Mol. Biol.
Source Genre: Journal
 Creator(s):
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Publ. Info: -
Pages: - Volume / Issue: 315 (3) Sequence Number: - Start / End Page: 447 - 457 Identifier: ISSN: 0022-2836