Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase

Abstract

Proteins obtain their final functional configuration through incremental folding with many intermediate steps in the folding pathway. If known, these intermediate steps could be valuable new targets for designing therapeutics and the sequence of events could elucidate the mechanism of refolding. 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 part of the folding pathway for the model large multidomain protein, Luciferase, by combining single-molecule force-spectroscopy experiments and coarsegrained simulation. Single-molecule refolding experiments reveal the initial nucleation of folding while simulations corroborate these stable core structures of Luciferase, and indicate the relative propensities for each to propagate to the final folded native state. Both experimental refolding and Monte Carlo simulations of Markov state models generated from simulation reveal 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 nonnative structures. We then engineer truncated variants of Luciferase whose sequences corresponded to the putative structure from simulation and we use atomic force spectroscopy to determine their unfolding and stability. These experimental results corroborate the structures predicted from the folding simulation and strongly suggest that they are intermediates along the folding pathway. Taken together, our results suggest that initial Luciferase refolding occurs along a vectorial pathway and also suggest a mechanism that chaperones may exploit to prevent misfolding.

Bacterial Hsp70 resolves misfolded states and accelerates productive folding of a multi-domain protein

“…Interestingly, rapid folding was also observed for the isolated N S -subdomain (FLuc190) (Fig. 3j, k), consistent with its ability to fold co-translationally 27,36 . This is in contrast to the slow folding of N S in the context of full-length FLuc (FLucN S ), which limits the folding yield (Fig.…”

“…9a). The type of inter-domain misfolding rescued by the KJE system is common in large multi-domain proteins 36,[51][52][53] and artificial polyproteins 21,35,54,55 , and might occur during de novo folding or upon heat stress-induced denaturation. Indeed, the KJE system has recently been shown to stabilize numerous multi-domain proteins against thermal denaturation in vivo (Fig.…”

Energetic dependencies dictate folding mechanism in a complex protein

“…S5), consistent with our finding that it does not help to stabilize domain III energetically or increase its folding rate. Interdomain misfolding is prevalent after complete unfolding of EF-G (16) and other multidomain proteins (15, 56, 57). Constant force refolding experiments at 3 pN indicate that off-pathway nonnative structures are also populated during simultaneous refolding of domains III, IV, and V, competing with and slowing down productive folding (Fig.…”

Mechanical Stability of a Small, Highly-Luminescent Engineered Protein NanoLuc

“…These processes underlie many important phenomena such as those related to cellular homeostasis [ 9 , 10 , 11 , 12 , 13 ], cancer metabolism [ 1 , 14 , 15 , 16 , 17 ], and the onset and treatment of protein misfolding diseases [ 1 , 18 , 19 , 20 ]. Single-molecule force spectroscopy (SMFS) techniques are particularly powerful tools for studying protein folding because they provide a means to directly apply forces to individual proteins under native conditions in order to measure their structural response and the internal forces that stabilize the protein [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , ...…”