NanoLuc tandem repeats show misfolding during thermal denaturation which is reversible by E. coli chaperones (DnaK/DnaJ/GrpE).

Abstract

Monomeric NanoLuc (Nluc) is a thermally robust bioluminescent protein with its thermal denaturation temperature at 58oC. In order to further examine this thermal behavior, we developed three protein constructs (monomeric, dyad, and triad Nluc). Interestingly, thermal denaturation experiments at 58oC showed sudden decrease in thermal stability when Nluc was linked to itself, dyad and triad Nluc constructs, while monomeric Nluc remained mostly folded under same conditions. Also, all three constructs maintained their activity (bioluminescence) when brought back to room temperature with no spontaneous recovery observed. However, when we examined the E. coli DnaK/DnaJ/GrpE chaperone system effect on this loss of thermal stability in refolding assays, we observed recovery in bioluminescence signal up to 70% for the poly-Nluc constructs. This observation strongly supports poly-Nluc is a strong chaperone substrate. Another interesting observation was that the refolding of these poly-Nluc constructs was possible at similar percentages even in the absence of GrpE showing a deviation from the canonical chaperone mechanism described up to now in literature. However, high GrpE concentrations were inhibitory to the protein refolding. Similarly, addition of disaggregase ClpB chaperone, showed no further assistance in refolding of the proteins and in some cases proved to be inhibiting the refolding. Lastly, we performed MD simulations of thermally unfolded and refolded monomeric and poly-Nluc constructs. These show that monomeric Nluc is able to correctly refold, while dyad Nluc misfolds with 78% and triad Nluc misfolds with 63%. Also, additional MD simulations suggest the reason of dyad and triad Nluc constructs misfolding is due to domain swapping preventing the correct refolding of these two constructs during thermal renaturation.