. Author manuscript; available in PMC: 2011 Mar 12.

Published in final edited form as: Biochem Biophys Res Commun. 2010 Feb 10;393(3):504–508. doi: 10.1016/j.bbrc.2010.02.035

Table 1. Characterization of Phosphomimetic Ser→Glu Mutants of P16.

Protein	IC₅₀ (nM)^a	Core Structure^b	ΔG_d^water (kcal/mol)^c	D_1/2 (M)^c	m (kcal mol^-1 M^-1)^c	Tm (°C)^d
P16 SSSS^e (WT)	72±15	Retained	2.32	0.72	3.23	46.0
P16 ESSS (S7E)	140±17	Retained	2.95	0.83	3.49	47.3
P16 SESS (S8E)	>750^f	Retained	2.14	0.70	3.05	46.1
P16 SSES (S140E)	114±21	Retained	2.49	0.79	3.07	41.7
P16 SSSE (S152E)	45±12	Retained	2.46	0.80	2.95	43.0
P16 AAAA	87±21	Retained	2.82	0.76	3.73	46.3

The estimated error in the determination of IC₅₀ is ± 20%, and a 3-fold change in the IC₅₀ values is regarded as biochemically significant [7].

Perturbations to the core structure of P16 were investigated as previously described [7, 13].

ΔG_d^water, D_1/2 (the denaturant concentration at the midpoint of transition), and m values were calculated according to a two-state transition model, and the error in ΔG_d^water was estimated to be ± 0.5 kcal/mol [9].

The error of Tm was estimated to be ± 0.5 °C [9].

Data cited from [13].

The exact IC₅₀ of P16 S8E was not determined simply because the maximum inhibition of CDK4 was not achieved in the presence of 2.0 μM P16 S8E, the highest concentration used in our assay.