Chaperones Rescue Luciferase Folding by Separating Its Domains

Scholl, Zackary N.; Yang, Weitao; Marszałek, Piotr E.

doi:10.1074/jbc.m114.582049

Cited by 32 publications

(22 citation statements)

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“…A second chaperone, secB, had a similar effect on tandem MBP oligomers, preventing stable aggregation, but secB bound primarily to the unfolded or molten-globule states, suppressing native folding as well 59 . A similar mechanism of action—preventing non-native inter-domain interactions—was also suggested by SMFS studies of the multi-domain protein luciferase 25 , as well as by fluorescence studies of huntingtin showing that the chaperone prefoldin suppresses the formation of toxic oligomers 60 .…”

Section: Discussionmentioning

confidence: 56%

“…Single-molecule approaches have been deployed successfully to study protein misfolding and aggregation, for example identifying misfolded states, determining misfolding pathways, detecting transient oligomeric intermediates and exploring the interactions stabilizing amyloid fibrils 7 16 17 18 19 20 21 22 . They have also started to be applied to unravel the mechanisms of molecular chaperones 23 , showing for example that chaperones help correct folding of substrate proteins by unfolding misfolded molecules to give them a new chance to refold, altering the folding rates of domains, and blocking tertiary contacts in the transition state 23 24 25 26 27 . However, there has been little single-molecule work to date on pharmacological chaperones, aside from studies of their effects on amyloid stability 22 .…”

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confidence: 99%

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Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

et al. 2016

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The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.

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Section: Discussionmentioning

confidence: 56%

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confidence: 99%

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

et al. 2016

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“…Understanding how these applied and destabilizing forces might affect not only the structure but catalytic activity of those proteins can provide new insights into the molecular mechanisms of how misfolding, aggregation, or recovery to their functional forms with the help of other chaperone proteins can contribute to cellular health or disease [ 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ]. However, SMFS with simultaneous measurement of catalytic activity of the protein to which forces are being applied has been rather challenging due to numerous technical difficulties [ 44 , 74 , 75 , 76 , 77 ].…”

Section: Introductionmentioning

confidence: 99%

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

Ding

Apostolidou

Marszałek

2020

IJMS

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NanoLuc is a bioluminescent protein recently engineered for applications in molecular imaging and cellular reporter assays. Compared to other bioluminescent proteins used for these applications, like Firefly Luciferase and Renilla Luciferase, it is ~150 times brighter, more thermally stable, and smaller. Yet, no information is known with regards to its mechanical properties, which could introduce a new set of applications for this unique protein, such as a novel biomaterial or as a substrate for protein activity/refolding assays. Here, we generated a synthetic NanoLuc derivative protein that consists of three connected NanoLuc proteins flanked by two human titin I91 domains on each side and present our mechanical studies at the single molecule level by performing Single Molecule Force Spectroscopy (SMFS) measurements. Our results show each NanoLuc repeat in the derivative behaves as a single domain protein, with a single unfolding event occurring on average when approximately 72 pN is applied to the protein. Additionally, we performed cyclic measurements, where the forces applied to a single protein were cyclically raised then lowered to allow the protein the opportunity to refold: we observed the protein was able to refold to its correct structure after mechanical denaturation only 16.9% of the time, while another 26.9% of the time there was evidence of protein misfolding to a potentially non-functional conformation. These results show that NanoLuc is a mechanically moderately weak protein that is unable to robustly refold itself correctly when stretch-denatured, which makes it an attractive model for future protein folding and misfolding studies.

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“…In the case of tandem repeat proteins, which exhibit minimal interactions between the native domains, high sequence identity among neighboring units increases their propensity to form off-pathway, misfolded structures (12, 13). Even though compact globular multidomain proteins are typically made up of dissimilar domains, their folding is severely hampered by interdomain misfolding (14–17). Assistance from molecular chaperones and cotranslational folding ensure efficient folding in the cell (18, 19).…”

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confidence: 99%

“…Dissecting multidomain protein folding and domain coupling is challenging (29), and few mechanistic studies of nonrepeat proteins are available to date (14–17, 30). Single-molecule force spectroscopy is a powerful tool for dissecting complex folding pathways (31).…”

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confidence: 99%

Energetic dependencies dictate folding mechanism in a complex protein

Liu

Chen

Kaiser

2019

Proc. Natl. Acad. Sci. U.S.A.

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Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

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Chaperones Rescue Luciferase Folding by Separating Its Domains

Cited by 32 publications

References 50 publications

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

Pharmacological chaperone reshapes the energy landscape for folding and aggregation of the prion protein

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

Energetic dependencies dictate folding mechanism in a complex protein

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