Reconstructing The Folding Of Luciferase To Elucidate The Vectorial Folding Pathways Of Large, Multidomain Proteins

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediates could be valuable new targets for designing therapeutics and could provide information on the mechanism of chaperones. However, determining these intermediate steps is hardly an easy feat, and has been elusive for most proteins, especially large, multidomain proteins. Here, we effectively map out the majority of a folding pathway for the model large multidomain protein, Luciferase, by combining coarse-grained simulation and single-molecule force-spectroscopy experiments. Simulations indicate that there are several consistent and stable core structures of various sizes nucleating in different regions of Luciferase, each of which has different propensities for propagating to the final folded native state. We identified, using Monte Carlo simulations of Markov chains generated from simulation, that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway is the least likely to form non-native structures. We engineered truncated variants of Luciferase whose sequences corresponded to the putative nucleated cores and using atomic force spectroscopy we determined their unfolding and stability. The experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. The simulation also identified non-native structures originating mainly from the C-terminal domain. Taken together, our results suggest a pathway for cotranslational folding of Luciferase and also suggest a mechanism that chaperones may exploit to prevent misfolding.