Figure 4.

Characterization of purified PH domain proteins. (A) Four different His6-GB1 fusion proteins were purified and run through a Sephadex S300 sizing column; wild type ItkPH and ItkPH(C96P/T110I) show signs of aggregation, eluting in two peaks, whereas ItkPH/TecHα and TecPH WT each elute as a single peak corresponding to the size of the monomeric PH domain. (B) Purified His6-GB1-ItkPH(C96P/T110I) and His6-GB1-ItkPH(C96E/T110I) were run through a Sephadex S100 sizing column. Unlike ItkPH(C96P/T110I), the ItkPH(C96E/T110I) mutant elutes as a single peak. (C) After cleavage of the His6-GB1 tag, purified ¹³C/¹⁵N-labeled ItkPH(C96E/T110I) was passed over a Superdex-75 column on an Aktä FPLC system (solid black line). SDS-PAGE analysis of the resulting fractions is shown above the FPLC trace. Protein standards (dashed purple and blue lines) are shown and indicate the major peak for ItkPH(C96E/T110I) elutes in a manner consistent with its 19 kDa monomeric molecular weight. After completion of NMR data acquisition, the IP₄-bound ItkPH(C96E/T110I) was re-injected onto the Superdex-75 column and run under identical conditions (solid red line). The slight shift in the elution profile of IP₄ bound ItkPH (compare red with black trace) is consistent with previously reported findings that PH domains exhibit restricted motions on binding to IP₄.³¹ (D) The two peaks (fractions 24–35 and 36–41) from the initial ItkPH(C96E/T110I) Superdex-75 run (black trace in (C)) were analyzed by CD spectroscopy. The two samples show no significant difference in secondary structure.