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. 2022 Jun 23;9:882288. doi: 10.3389/fmolb.2022.882288

FIGURE 8.

FIGURE 8

hENTH forms complexes in micellar concentrations of PI(4,5)P2. (A) nDSF transitions for hENTH at 30 µM in the absence and presence of 200 µM PI(4,5)P2. The shift in the sample with PI(4,5)P2 indicates that the oligomer unfolds before the monomers. (B) Scattering curves of hENTH in the absence and presence of 200 µM PI(4,5)P2. The hENTH aggregates at lower temperature in the presence of PI(4,5)P2. (C) DLS auto-correlation curves of hENTH at 30 µM in the absence and presence of 200 µM PI(4,5)P2. The shift in the autocorrelation curve indicates that the hENTH domain oligomerizes in the presence of PI(4,5)P2. (D) Histogram of the masses present on the DLS samples in the absence and presence of PI(4,5)P2. hENTH shifts towards higher masses in the presence of PI(4,5)P2, consistent with oligomer formation. (E) hENTH at 100 nM (monomer MW = 18.5 kDa) shows a peak at 108 kDa, corresponding to a hexamer of hENTH in the presence of 200 µM PI(4,5)P2 at 112 kDa. (F) Control: mass photometry histogram of buffer with 200 µM PI(4,5)P2. A peak at ca. 51 kDa corresponds to PI(4,5)P2 micelles. (G) Schematic of the hENTH domain (green) (PDB ID: 5ONF) and the oligomer model with PI(4,5)P2 molecules (yellow) from Garcia-Alai et al., 2018. (H) Representative negative staining and cryo-electron micrograph of hENTH + PI(4,5)P2 where hENTH hexamers can be observed. Particles of interest are indicated with white arrows.