Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex

Figures

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  Figure 1. Size exclusion chromatography of His6 tagged VPg, His6 tagged 4E (eIF4E) and His6 VPg-His6 eIF4E, their binary complex. (A) Separated runs of the purified monomers (1 mL of 0.7–1 mg/mL) were performed. Vertical dashed lines refer to the elution volumes expected for globular proteins of 28,549 Da (upper panel) and 26,137 Da (middle panel), the molecular weights of recombinant His6 eIF4E and His6 VPg respectively (MALDI-TOF spectrometry determinations). The His6 VPg-His6 eIF4E binary complex was pre-formed by mixing His6 eIF4E (0.7 mg/mL with an excess of His6 VPg (1.2 mg/mL) and loaded up to the column (lower panel). A mass of 54,772 Da was estimated for the binary complex (summing His6 VPg and His6 eIF4E molecular weights) vertical dashed line. For comparison, absorbance values were standardized to the maximum value of each peak. (B) Distribution of the various molecular species through the size exclusion chromatography (SEC) separation of a His6 VPg-His6 eIF4E mix. Upon elution, fractions 1. 2 and 3 were recovered and submitted to SDS-PAGE analysis. (C) Determination of the three molecular species apparent molecular weights (Mapp) deduced from standard calibration with a set of…

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  Figure 2. Fluorescence anisotropy of the various molecular forms of eIF4E and VPg. (A) The fluorescence anisotropy of His6 VPg* (300 nM, open circles) and His6 VPg*-His6 eIF4E complex (mix of 300 nM His6 VPg* and 2 µM His6 eIF4E filled circles) was recorded as a function of the viscosity increase at 25 °C. The reciprocal of the emitted light anisotropy (Perrin’s plot), 1/A, is plotted as a function of T/η. V, the apparent molecular volume of the proteins and their complexes and A0, the fundamental anisotropy were obtained respectively from the slope and intercept at infinite viscosity. Inset, the fluorescence anisotropy increase upon association of His6 eIF4E with His6 VPg* (300 nM). (B) Perrin’s plots of untagged VPg* (open circles) and VPg*-eIF4E complex (filled circles) in the same conditions as (A). (C) Perrin’s plots of His6 VPg* (open squares), VPg* (filled circles) and D. His6 eIF4E* (filled circles), eIF4E* (open squared) and eIF4EΔ1–46* (filled triangles). Experimental conditions were the same as in B. All measurements were obtained using VPg from lettuce mosaic virus (LMV) AF199 strain.