The Hsp70 chaperone system performs a critical function in the maintenance of protein homeostasis and cellular health. One such important role is the folding of nascent proteins from a polypeptide chain at synthesis or refolding proteins that have engaged in incorrect folding pathways. This refolding action is thought to occur by remodelling of the client protein by the chaperone machinery; however, many of the specific molecular interactions that underpin this process have yet to be well characterised. This study aims to interrogate these processes through the use of single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments within a Total Internal Reflection Fluorescence (TIRF) framework. Double-cysteine mutants of firefly luciferase (Fluc) that could be site-specifically labelled with fluorophores were generated to act as model client proteins for chaperone machinery. In concert with enzymatic luminescence assays, smFRET experiments are to be performed to monitor the conformational changes undergone by these clients as they interact with the mammalian Hsp70 system (HspA8, DnaJA2, Hsp110). We aim to resolve mechanistic details regarding the number of cycles of Hsp70 binding and release required for productive refolding, as well as any conformational remodelling of the client protein. Additionally, by using the different Fluc mutant constructs, we hope to report on how different regions within the protein structure fold, misfold and refold in the absence and presence of molecular chaperones.