Gradual Folding of an Off-Pathway Molten Globule Detected at the Single-Molecule Level

Lindhoud, Simon; Pirchi, Menahem; Westphal, Adrie H.; Haran, Gilad; Mierlo, C.P.M. van

doi:10.1016/j.jmb.2015.07.002

Cited by 18 publications

(33 citation statements)

References 64 publications

(107 reference statements)

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“…This typical feature probably contributes to the observed misfolding, as upon folding the α-helices fold first and can subsequently dock onto one another through hydrophobic interactions. This docking prevents formation of the parallel β-sheet and the resulting misfolded intermediate needs to unfold considerably before folding to native protein can take place, explaining why this species is off-pathway, as experimentally shown for A. vinelandii flavodoxin (67,(70)(71)(72)76,78). Indeed, this off-pathway species is largely α-helical (69).…”

Section: Flavodoxin-like Proteins Tend To Temporarily Misfold During mentioning

confidence: 93%

“…Its off-pathway nature is due to docking of non-native α-helices, thereby preventing formation of the central β-sheet of native flavodoxin (Fig. 4) (70, 71,78). Formation of off-pathway folding intermediates seems to be typical for proteins with a flavodoxin-like fold, as they have also been observed for Anabaena apoflavodoxin and chemotactic protein (59,(79)(80)(81).…”

Section: Elucidating Cotranslational Folding Of Flavodoxinmentioning

confidence: 94%

“…This mutation de facto introduces an oxygen atom into a hydrophobic pocket of apoflavodoxin and destabilizes its native state (78,82). F44Y apoflavodoxin is thus an interesting candidate to study formation of off-pathway MGs in vivo, as substantial population of MG off occurs at physiologically relevant conditions.…”

Section: Elucidating Cotranslational Folding Of Flavodoxinmentioning

confidence: 99%

“…While formation of native apoflavodoxin is highly cooperative (5,61,64,78), conversion of unfolded protein into MG off is a non-cooperative, gradual process that simultaneously involves separate regions within apoflavodoxin (70,78). This suggests an energy landscape of MG folding with many barriers (visualised by shallow moats in the off-pathway trough of Fig.…”

Section: Scheme Bmentioning

confidence: 99%

“…4). In vitro, folding of the C-terminal part of MG off precedes folding of the N-terminal part and this intermediate gradually compacts due to progressive extension of its ordered core (76,78). This non-cooperative folding happens because helices involve relatively short-range interactions and the folding of one helix does not need to affect the folding of other helices (70).…”

Section: Scheme Bmentioning

confidence: 99%

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The ribosome regulates flavodoxin folding

Houwman¹

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Section: Flavodoxin-like Proteins Tend To Temporarily Misfold During mentioning

confidence: 93%

Section: Elucidating Cotranslational Folding Of Flavodoxinmentioning

confidence: 94%

Section: Elucidating Cotranslational Folding Of Flavodoxinmentioning

confidence: 99%

Section: Scheme Bmentioning

confidence: 99%

Section: Scheme Bmentioning

confidence: 99%

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The ribosome regulates flavodoxin folding

Houwman¹

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A look at the face of the molten globule: Structural model of the Helicobacter pylori apoflavodoxin ensemble at acidic pH

et al. 2022

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Molten globule (MG) is the name given to a compact, non‐native conformation of proteins that has stimulated the imagination and work in the protein folding field for more than 40 years. The MG has been proposed to play a central role in the folding reaction and in important cell functions, and to be related to the onset of misfolding diseases. Due to its inherent intractability to high‐resolution studies, atomistic structural models have not yet been obtained. We present here an integrative atomistic model of the MG formed at acidic pH by the apoflavodoxin from the human pathogen Helicobacter pylori. This MG has been previously shown to exhibit the archetypical expansion, spectroscopic and thermodynamic features of a molten conformation. To obtain the model, we have analyzed the stability of wild‐type and 55 apoflavodoxin mutants to derive experimental equilibrium Φ values that have been used in biased molecular dynamics simulations to convert the native conformation into an MG ensemble. The ensemble has been refined to reproduce the experimental hydrodynamic radius and circular dichroism (CD) spectrum. The refined ensemble, deposited in PDB‐Dev, successfully explains the characteristic 1H‐nuclear magnetic resonance (NMR) and near‐UV CD spectral features of the MG as well as its solvent‐accessible surface area (SASA) change upon unfolding. This integrative model of an MG will help to understand the energetics and roles of these elusive conformations in protein folding and misfolding. Interestingly, the apoflavodoxin MG is structurally unrelated to previously described partly unfolded conformations of this protein, exemplifying that equilibrium MGs need not to reflect the properties of kinetic intermediates.

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