As a delicate origami figure loses its shape in the wind, proteins, the essential building blocks of life, can unfold in the chaotic, crowded, and mechanically dynamic environment of the cell. These structures must be constructed in a precise three-dimensional manner to function, yet the cellular interior constantly threatens that order. To preserve structure and function, cells utilize highly specialized molecular machines known as chaperones, which fold, refold, and, if necessary, dismantle proteins with exceptional precision. Understanding how these machines work at the molecular level is key to understanding how life resists disorder and maintains function despite relentless internal chaos.

Chapter 1, is a review that describes how single-molecule techniques have advanced our understanding of chaperone-substrate interactions, while also highlighting key limitations that call for new experimental strategies to fully resolve their dynamic and heterogeneous behavior.

In Chapter 2, we examined the bacterial Hsp70 chaperone system, comprising DnaK, its cochaperone DnaJ, and the nucleotide exchange factor GrpE. As a model substrate, we used a slow-folding variant of the Escherichia coli maltose-binding protein (MBP). We found that this system accelerates the folding of otherwise slow-folding proteins. Using optical tweezers, we tracked the conformational transitions of an unfolded maltose-binding protein variant. We discovered that DnaK, upon ATP-driven cycling, induces a compacted, dynamic collapse state that primes the protein for rapid folding. This compaction is crucial for overcoming energy barriers and is a previously neglected active function of Hsp70: beyond shielding, it changes the folding process itself. Our findings demonstrate that the Hsp70 system is not just a passive chaperone, but actively affects folding pathways, with mechanical signatures that can be resolved in single-molecule detail.

In Chapter 3, we examined the archaeal Group II chaperonin CpN from Methanococcus maripaludis (mmCpN), focusing on its role in mechanically assisted folding of a model substrate protein (Rhodanese). The findings highlight how mmCpN contributes to distinct mechanical functions: stabilizing intermediates and enabling transitions to the native state. Wild-type mmCpN promoted large-scale folding events and stabilized force-resistant intermediates, while a C-terminal truncation mutant retained intermediates but failed to support productive folding. This revealed that mmCpN transforms the energy landscape, with different domains contributing to stabilization and folding. These findings refine our understanding of chaperonin modularity and mechanical assistance in folding under force. These experiments demonstrate how structural modules within the same protein can partition distinct biophysical functions: one domain anchoring, another driving structural transitions, highlighting the modular engineering principles behind complex molecular machines.

Chapter 4 shifts focus on innate immunity and the role of the human GTPase GBP1. Guanylate-binding protein 1 (GBP1) is a dynamin-like GTPase involved in human cells' interferonmediated immune response. We utilized a dual-trap optical tweezer assay in combination with confocal fluorescence imaging to visualize membrane remodeling events in real time. Our data demonstrates that GBP1-mediated scission is dependent on GTP hydrolysis, indicating the existence of a mechanical cycle that actively disrupts membranes. These findings suggest that GBP1 is a forcex generating immune effector that converts nucleotide energy into membrane destabilization, a powerful mechanism in host defense.

Together, this work shows the diverse strategies cells employ to guide structure formation or disassembly—from polypeptide collapse to membrane scission. Each system converts chemical energy into mechanical influence, revealing how ATP- and GTP-consuming machines promote biological order. This work not only advances our understanding of protein homeostasis, but also may be beneficial for future efforts to engineer or therapeutically target molecular machines at the nanoscale. By integrating high-resolution force spectroscopy with biochemical and structural perspectives, this thesis provides a molecule-by-molecule analysis of how the living cell physically manipulates its inner world.