Date

Thursday 03 Sep 2026

Time

15:15 - 15:45

Location

EM119

Supervisor

Anne Wentink

2nd reviewer

Anjali Pandit

Jury

Francesco Buda

J-domain proteins (JDPs) function as essential co-chaperones for the Hsp70 system by delivering substrates and stimulating ATP hydrolysis. In JDPs involved in amyloid clearance, molecular autoinhibition regulates function via a regulatory helix that docks onto the Hsp70 binding site, preventing premature Hsp70 activation. However, the precise structural mechanisms that control how this autoinhibition is relieved to trigger amyloid clearance remain poorly understood. In this study, we engineered and characterized novel double-cysteine DNAJB1 variants to elucidate the structural mechanisms controlling J-domain release and JDP activity. Using functional assays, we demonstrated that the double-cysteine mutant (DNAJB1 G40C G194C) still retained its ability to stimulate Hsp70 ATPase activity and effectively disaggregate α-synuclein fibers. Conversely, a regulatory helix-deficient variant maintained ATPase stimulation but failed to disaggregate amyloid fibers, validating the belief that this structural element is required for the disaggregation of amyloids. To monitor these conformational changes, we developed a FRET-based assay. With this assay, the structural state of DNAJB1 upon the addition of potential J-domain releasing factors such as Hsp70, the C-terminal EEVD peptide, or α-synuclein fiber can be investigated. Although the results of this assay were inconclusive, this work still establishes a robust framework for using FRET to map the conformational changes during DNAJB1 autoinhibition. Optimizing this preliminary FRET framework will ultimately allow us to uncover the precise molecular triggers behind J-domain release, advancing our broader understanding of the unique functional diversity within the J-domain protein family.