title: Protein folding and stability of human CDK inhibitor p19(INK4d).
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 (1)H-(15)N 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.
authors.affiliation: Laboratorium für Biochemie, Universität Bayreuth, D-95440 Bayreuth, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Zeeb
authors.forename: Heike
authors.initials: H
authors.lastname: Rösner
authors.forename: Wojciech
authors.initials: W
authors.lastname: Zeslawski
authors.forename: Denis
authors.initials: D
authors.lastname: Canet
authors.forename: Tad A
authors.initials: TA
authors.lastname: Holak
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
mesh_terms: D018797:Cell Cycle Proteins; D002942:Circular Dichroism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000737:chemistry* / Q000378:metabolism*; D050764:Cyclin-Dependent Kinase Inhibitor p19; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010649:Phenylalanine / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
chemical_list: C493974:CDKN2D protein, human; D018797:Cell Cycle Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050764:Cyclin-Dependent Kinase Inhibitor p19; D010649:Phenylalanine; D014508:Urea; D018844:Cyclin-Dependent Kinases