Fig. 7

Design and characterization of the membrane binding-deficient K18 and Tau mutants. a Helical wheel and β-strands depictions to illustrate the rational for the design of the K18 and Tau mutants. Mutations are in purple, hydrophilic residues in blue, hydrophobic residues in black, positively charged residues in red, cysteine in green, and glycine in white. The hydrophobic and hydrophilic faces of the helices are depicted in light gray and blue, respectively, and the putative position of the negatively charged phospholipid head groups are indicated by dashed lines. Mutation of a hydrophobic Val residue in the hydrophobic part of an amphipathic motif to a negatively charged Glu should interfere with the capacity of the segment to embed itself into the hydrophobic core of a membrane. In the case of Tau, the modification of positively charged Lys-311 to an uncharged Ala should affect its capacity to interact with negatively charged membranes through electrostatic interactions. b Co-sedimentation assay of BPS vesicles with WT and MBD-K18 (top) and Tau (bottom). When incubated alone (left), all proteins remain in the supernatant (sup.), but in the presence of BPS vesicles (right), the proteins partially co-sediment with the vesicles to the pellet (pel.), indicative of membrane binding. Compared with the WT proteins, more MBD-K18 and MBD-Tau proteins remain in the supernatant, indicating that they partially lost the ability to bind membranes. c Top: size-exclusion chromatograms of WT K18 (solid lines) and MBD-K18 (dashed lines) alone (blue lines) or with BPS vesicles (red lines) show that only WT K18 forms protein/phospholipid complexes. Bottom: size-exclusion chromatograms of WT Tau (blue line) and MBD-Tau (red line) show that WT Tau is efficient in forming protein/phospholipid complexes (blue, large peak) while the capacity of MBD-Tau to form such complexes is compromised (red, small peak). d EM analysis of WT and MBD-K18 (top) and Tau (bottom) shows that the mutants cannot form protein/phospholipid complexes in the presence of BPS vesicles (second-to-left panels) but retain their ability to form fibrils (right-most panels). The scale bars are 100 nm