Quantitative NMR analysis of the mechanism and kinetics of chaperone Hsp104 action on amyloid-& beta;42 aggregation and fibril formation

The chaperone Hsp104, a member of the Hsp100/Clp family of translocases, prevents fibril formation of a variety of amyloidogenic peptides in a paradoxically substoi-chiometric manner. To understand the mechanism whereby Hsp104 inhibits fibril formation, we probed the interaction of Hsp104 with the Alzheimer's amyloid-(342 (A(342) peptide using a variety of biophysical techniques. Hsp104 is highly effective at suppressing the formation of Thioflavin T (ThT) reactive mature fibrils that are readily observed by atomic force (AFM) and electron (EM) microscopies. Quantitative kinetic analysis and global fitting was performed on serially recorded 1H -15N corre-lation spectra to monitor the disappearance of A(342 monomers during the course of aggregation over a wide range of Hsp104 concentrations. Under the conditions employed (50 & mu;M A(342 at 20 & DEG;C), A(342 aggregation occurs by a branching mecha-nism: an irreversible on-pathway leading to mature fibrils that entails primary and secondary nucleation and saturating elongation; and a reversible off-pathway to form nonfibrillar oligomers, unreactive to ThT and too large to be observed directly by NMR, but too small to be visualized by AFM or EM. Hsp104 binds reversibly with nanomolar affinity to sparsely populated A(342 nuclei present in nanomolar concentrations, generated by primary and secondary nucleation, thereby completely inhibiting on-pathway fibril formation at substoichiometric ratios of Hsp104 to A(342 monomers. Tight binding to sparsely populated nuclei likely constitutes a general mechanism for substoichiometric inhibition of fibrillization by a variety of chaperones. Hsp104 also impacts off-pathway oligomerization but to a much smaller degree initially reducing and then increasing the rate of off-pathway oligomerization.

Automated summary

The chaperone Hsp104 prevents fibril formation of amyloidogenic peptides in a substochiometric manner. By studying its interaction with the A(342) peptide, it was found that Hsp104 binds reversibly to sparsely populated nuclei of A(342), completely inhibiting fibril formation.