Utilising Fluorescence Microscopy to Visualise the Dynamics and Interactions of Molecular Chaperones and α-Synuclein

Abstract

Cellular protein quality control comprises a network of chaperones that maintain the proteome viability by performing key cellular tasks such as degrading or remodeling misfolded proteins. Bacterial Caseinolytic proteases (Clp) which are responsible for protein degradation include powerful ring-shaped AAAþ (ATPases Associated with diverse cellular Activities) motors with a central narrow pore that unfold and translocate tagged abnormal proteins. Clp ATPase machines thread substrate proteins (SPs) through their central channel by using repetitive ATP-driven subunit motions coupled with axial mechanical forces exerted onto the SP. Here we perform multiscale molecular simulations of ClpYDI and Titin I27 to mimic and contrast laser optical tweezer (LOT) experiments, in which the SP N-terminus is restrained, with in vivo ClpY-mediated unfolding and translocation, in which the SP is not restrained at the N-terminus. This allows us to shed light on the effects of restraining forces and SP mechanical direction probed on Clp-mediated unfolding mechanism. The external LOT restraint limits ClpY-mediated pulling along the N-C direction of the SP, which yields unfolding of I27 via a shearing mechanism. By contrast, in vivo-like ClpY-action results in pulling along softer mechanical directions and I27 is unfolded via an unzipping mechanism. We find that factors that affect these distinct mechanisms are SP-ClpY surface interactions, the size of the SP relative to the ClpY pore size, the SP mechanical resistance, and the presence of other substrate domains.