FIGURE 5.

Deletion of amino acids 129–148 diminishes functional interactions with Hsp104. A, the ATPase activity of Hsp104 was measured in the presence of Md and MdΔ129–148 and expressed as the fold-increase over activity measured in the absence of peptide. B, the fibrilization of 3 μm NM in the presence of Hsp104, huHsp70, and Ydj1 (0.6 μm monomers each) was monitored in the presence of increasing concentrations of MdΔ129–148. The enhancement of acrylodan fluorescence was normalized to the maximum value obtained in reactions without addition of peptide. C, the binding of Hsp104^Trap to fluorescently labeled Md and MdΔ129–148 was measured using fluorescence anisotropy in the presence of ATP. D, spontaneous and chaperone-dependent fibrilization of intact NM and NMΔ129–148 were compared. For spontaneous fibrilization, 3 μm of the each protein was rotated overnight and fibrilization was measured by ThT fluorescence and expressed as the fold-increase in fluorescence in samples compared with the starting material. Chaperone-dependent reactions were as described in panel B but ThT fluorescence was used to determine the extent of fibril formation. E, comparison of fibrils produced from NM and NMΔ129–148 seeded with preformed NM fibrils using negative stain TEM (inset) and thermal stability of fibrils. Top panels show Coomassie-stained gels of proteins entering the gel after heating at the indicated temperature in SDS. Lower panel shows quantitation of solubility.