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. Study holds ProTherm entries: 13026, 13027 Extra Details: p19(INK4d); protein folding intermediate; protein folding; NMR;,phenylalanine fluorescence
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||2|
|Proteins||Cyclin-dependent kinase 4 inhibitor D ; Cyclin-dependent kinase 4 inhibitor D|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: Tm|
|Libraries||Mutations for sequence RAGDRLSGAAARGDVQEVRRLLHRELVHPDALNRFGKTALQVMMFGSTAIALELLKQGASPNVQDTSGTSPVHDAARTGFLDTLKVLVEHGADVNVPDGTGALPIHLAVQEGHTAVVSFLAAESDLHRRDARGLTPLELALQRGAQDLVDILQGHM|