Chaperone-mediated protein folding and rescue

This thesis investigates how cells use energy-driven molecular machines to maintain
order in a crowded and mechanically active environment where proteins can misfold or
unfold.

Chapter 1 reviews how single-molecule methods have advanced our understanding of
chaperone–substrate interactions, while identifying key limitations that still hinder full
resolution of their dynamic and heterogeneous behavior.

Chapter 2 focuses on the bacterial Hsp70 system (DnaK, DnaJ, and GrpE) using a slowfolding maltose-binding protein variant as a model substrate. Optical tweezers experiments show that ATP-driven DnaK cycling induces a compact, dynamic collapse
state that primes the unfolded protein for rapid folding. This reveals an active role for Hsp70 beyond passive shielding: it reshapes the folding pathway itself by helping overcome energy barriers.

Chapter 3 examines the archaeal Group II chaperonin mmCpN and its role in folding
rhodanese under force. Wild-type mmCpN promotes large folding transitions and
stabilizes force-resistant intermediates, whereas a C-terminal truncation mutant
stabilizes intermediates but fails to support productive folding. These results show that
distinct structural domains of mmCpN contribute separately to stabilization and folding,
highlighting functional modularity.

Chapter 4 investigates the human immune GTPase GBP1 using dual-trap optical
tweezers and confocal fluorescence imaging. The data show that GBP1-mediated
membrane scission depends on GTP hydrolysis, supporting a mechanical cycle that
actively destabilizes membranes.

Overall, this thesis demonstrates how ATP- and GTP-driven machines convert chemical
energy into mechanical work to control protein folding and membrane remodeling at the
single-molecule level.