Mechanical and thermal stability of tandem repeats of highly-bioluminescent protein NanoLuc

Proteins are molecules found in all living kingdoms due to their several cellular functions. They are comprised of amino acids, which are linked together forming long polypeptide chains. These chains can fold into specific three dimensional structures, unique to every protein, which are directly related to their function. However, proteins can lose this structure for various reasons (e.g., increase of temperature). This can result in protein misfolding and possibly formation of aggregates that are dangerous to cell viability. Some of these aggregates are known to result in diseases, such as Alzheimer's disease.Additionally, in cells there is a class of proteins, the molecular chaperones. Those have been shown to assist other proteins to fold into their native state, along with assist in breaking down aggregates to prevent cell death. However, the mechanisms in which chaperones are able to do so, are not fully understood and are still under investigation. Up till now, only a small number of proteins have been used in the chaperones mechanism studies, despite the large number of substrate proteins they assist. Therefore, it is likely that our current view of the chaperones mechanism may be limited with regards to their overall mechanism. At the same time, with the recent developments in the biotechnology industry, understanding and predicting functional protein folding structures has had a great impact in the development of therapeutic proteins. Here, we aim to examine both the protein unfolding and refolding of a newly identified protein, NanoLuc (Nluc). Nluc is a highly bioluminescent protein, despite its small 19 kDa size. In comparison, Nluc is approximately 3 times smaller than firefly luciferase (Fluc, 61 kDa), a protein that has been extensively used in molecular chaperone assisted refolding studies. However, Nluc produces 150 times brighter bioluminescence signal to Fluc. Additionally, Nluc bioluminescence reaction is ATP independent, unlike Fluc who requires ATP energy to produce the bioluminescence signal. This bioluminescence signal, regardless of the protein, is directly related to the protein’s native state. Thus, any structural compromise in Nluc due to denaturing conditions will result in loss of its bioluminescence signal in bioluminescence assays. Combining this with chaperone proteins, we aim to examine how chaperones assist the refolding of thermally denatured Nluc. In this way we aim to introduce various substrates of Nluc repeats with the goal of identifying new possible chaperone and substrate interactions. Additionally, we engineered novel poly-protein constructs with tandem repeats of Nluc and titin I91 domains and performed mechanical unfolding and misfolding experiments. The aim behind these experiments was to examine the misfolding and refolding behavior of Nluc when it is linked to itself versus when it is separated by the titin I91 domains. Our findings showed that the unfolding behavior of the various constructs were very similar to one another, however their refolding was different. We observed via cyclic mechanical unfolding/refolding studies that when Nluc proteins were separated with titin I91 domains, the protein was less likely to misfold, than when the Nlucs were linked next to each other. A more detailed description per chapter is provided below. In chapter one, we introduce basic principles of biochemistry and physics to be later used. Those include defining what is a protein, introducing thermodynamic laws, discussing the protein folding paradox and the energy landscapes of proteins, and finishing with an introduction to molecular chaperones. In chapter two, we introduce the materials and methods used in this work. We start with explaining what bioluminescence is. Then, we proceed with introducing Nluc along with the engineered protein constructs used in this work. We describe our detailed protocols on protein expression and purification. A description of our bioluminescence protocol is also provided. Then we proceed with an introduction to the Atomic Force Microscopy (AFM)-based Single Molecule Force Spectroscopy (SMFS) technique, which relies on the elastic properties of the polypeptide chain of proteins. Worm-like chain model is explained along with basic operation of the device used during the experiments. Lastly, we introduce Molecular Dynamic (MD) Simulations, and we present the protocol followed in those simulations. In chapter three, we present the AFM-based SMFS experiments for one of the Nluc protein constructs, ?912−????3−?912. In this chapter we examine and discuss the unfolding behavior of this poly-Nluc and titin I91 domain construct in which the three Nlucs are linked next to each other by a very short linker (5 amino acids). The aim is to understand how linking the Nluc repeats in close proximity affects their misfolding behavior. In chapter four, similarly to chapter three, we present the AFM-based SMFS experiments for two of the engineered Nluc protein constructs, ?912−????−?914 and ?91−????−?91−????−?91−????−?91. In one of these constructs, we have the same number of Nluc proteins (three) and titin I91 domains (four), as the construct in chapter three, but in this construct the Nlucs are separated from each other by titin I91 domains. By mechanically unfolding/refolding this construct, we can examine how separating the various Nlucs affects their unfolding and refolding behavior. We also examined the unfolding and refolding behavior of a single Nluc flanked by titin I91 domains. The two constructs in this chapter were compared in order to examine if the three Nlucs behaved as three “single” Nlucs, like in the ?912−????−?914 construct. Our findings showed they had similar behavior. Lastly, we present results on the simulations for steered MD simulations along with coarse-grained MD simulations. The coarse-grained MD simulations were performed by Dr. Pan Zhang, a PhD candidate at that time in Dr. Weitao Yang’s group as part of a collaboration. In chapter five, we tested three protein constructs we engineered that we used in our chaperone assisted refolding studies via bioluminescence assays. The constructs generated were monomeric, dyad, and triad Nluc, which we thermally denatured at the melting temperature of 58?? in the absence of chaperones. During our experiments we observed that monomeric Nluc demonstrated strong thermal stability (minimal loss in bioluminescence signal), as previously reported in literature. However, dyad and triad Nluc constructs would lose their bioluminescence signal indicating a loss in their native three-dimensional conformation. Therefore, we performed chaperone assisted refolding studies to examine the recovery of these proteins, and we observed both were strong chaperone substrates. Another interesting observation was the fact that GrpE, a known nucleotide exchange factor of DnaK, did not have a significant effect in refolding of these constructs. This is contrary to the literature up till now as in most cases GrpE is shown to be very important in the refolding of substrate proteins, e.g., in Fluc refolding assays, as we observed in our control experiments. Additionally, we performed MD simulations to try and elucidate possible misfolding of the various Nluc constructs. We performed both coarse-grained simulations (by our collaborator Dr. Pan Zhang and Dr. Weitao Yang) and all-atoms simulations. Lastly, we present the TADOSS domain swapping simulations results from our collaborators (Dr. Pan Zhang and Dr. Weitao Yang) to provide some computational understanding to possible misfolding events of poly-Nluc constructs. In chapter six, we conclude our findings and present future work on our study.