Topological Switching between an α−β Parallel Protein and a Remarkably Helical Molten Globule

Nabuurs, Sanne M.; Westphal, Adrie H.; Toorn, Marije aan den; Lindhoud, Simon; Mierlo, C.P.M. van

doi:10.1021/ja9014309

Cited by 14 publications

(58 citation statements)

References 49 publications

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“…We note that the apo form of − 5 P is more susceptible to degradation than apo-FL P , which is in agreement with our observation that site-directed mutagenesis of apoflavodoxin decreases the stabilities of the corresponding native apo-proteins [38,45,51,52]. In addition, flexibility of the flavin-binding site in flavodoxin is much lower than in apoflavodoxin and holoprotein is much more stable than apo-protein [25,53,54].…”

Section: Released Fl P and −5 P Contain Fmn And Are Natively Foldedsupporting

confidence: 91%

“…Apoflavodoxin molecules that are unable to incorporate FMN explain why we previously observed less over-production in E. coli (strain TG2) of protein variants of flavodoxin (like e.g. F44Y, W128F, W167F and W128F/W167F [38,56]) compared to wild-type protein. During the 12 to 20 h lasting over-production of these protein variants, cells have to produce a large amount of FMN to satisfy the requirements for cofactor.…”

Section: Discussionmentioning

confidence: 83%

“…This species is off the productive folding pathway and needs to unfold to embark on a route to native protein [36]. The molten globule consists of a loosely packed core and solvent exposed hydrophobic side chains [37][38][39]. For many proteins with an α/β-parallel topology, formation of an off-pathway intermediate seems to be a characteristic feature [40].…”

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

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Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Houwman

Westphal

Berkel

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent flavodoxin, by generating ribosome-arrested nascent chains that expose either the entire protein or C-terminally truncated segments thereof. The native α/β parallel fold of flavodoxin is among the most ancestral and widely distributed folds in nature and exploring its co-translational folding is thus highly relevant. In Escherichia coli (strain BL21(DE3) Δtig::kan) FMN turns out to be limiting for saturation of this flavoprotein on time-scales vastly exceeding those of flavodoxin synthesis. Because the ribosome affects protein folding, apoflavodoxin cannot bind FMN during its translation. As a result, binding of cofactor to released protein is the last step in production of this flavoprotein in the cell. We show that once apoflavodoxin is entirely synthesized and exposed outside the ribosome to which it is stalled by an artificial linker containing the SecM sequence, the protein is natively folded and capable of binding FMN.

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Section: Released Fl P and −5 P Contain Fmn And Are Natively Foldedsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 83%

Section: Contents Lists Available At Sciencedirectmentioning

confidence: 99%

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Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Houwman

Westphal

Berkel

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…The molten globule is thus relatively compact. Far-UV CD reports that the molten globule contains helices (34). Fluorescence anisotropy of the tryptophans of the molten globule equals a value of 0.08, whereas fluorescence anisotropy of unfolded flavodoxin in GdnHCl is 0.05 (34).…”

Section: Discussionmentioning

confidence: 99%

“…Use is made of Tyr 44 -flavodoxin, in which Phe 44 is substituted for Tyr 44 . This substitution leads to severe destabilization of native apoflavodoxin against unfolding and only marginally affects the stability of the molten globule (34). As a consequence, Tyr 44 -apoflavodoxin forms the molten globule at low salt concentration (i.e.…”

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confidence: 99%

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

Nabuurs

Mierlo

2010

Journal of Biological Chemistry

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Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. thus form the stable core of the helical molten globule of ␣-␤ parallel flavodoxin, which is almost entirely structured. Nonnative docking of helices in the molten globule of flavodoxin prevents formation of the parallel ␤-sheet of native flavodoxin. Hence, to produce native ␣-␤ parallel protein molecules, the off-pathway species needs to unfold.Non-native protein conformations initially drew attention by their importance for the understanding of the process of protein folding (1-3). Now it is recognized that these conformations also yield important information about protein misfolding and aggregation (4). Partially folded states of proteins with exposed hydrophobic surfaces, such as molten globules, are prone to aggregation and can be precursors of amyloid fibril formation. This aggregation phenomenon can have devastating effects on organisms (4). The resemblance between early kinetic intermediates and molten globules (5-8) suggests that these molten globules can be considered as models of transient intermediates (9). This resemblance has been demonstrated for ␣-lactalbumin (10 -12), apomyoglobin (13,14), RNase H (15), T4 lysozyme (16), Im7 (17), and flavodoxin (18). Understanding the formation and conformation of these molten globules offers insights into factors responsible for protein misfolding and, potentially, for numerous debilitating pathologies (19).Upon descending the folding funnel, proteins encounter folding energy landscapes that are rough (20, 21). As a result, partially folded intermediates, which may be on-or off-pathway to the native state, are populated. When the intermediate is on-pathway, as is observed for the majority of proteins studied to date, it has a native-like topology and is productive for folding. In contrast, when the intermediate is off-pathway, it is trapped in such a manner that the native state cannot be reached without substantial reorganizational events (9). Several kinetic studies have revealed involvement of off-pathway intermediates during protein folding (18,22,23). A decrease of the folding rate due to the presence of an off-pathway molten globule, which is kinetically trapped and partially folded, increases the likelihood of protein aggregation.Here, using H/D exchange 2 experiments, we report the characterization of the off-pathway molten globule folding intermediate of a 179-residue flavodoxin from Azotobacter vinelandii. Flavodoxins are monomeric proteins involved in electron transport and contain a non-covalently bound FMN cofactor. The proteins consist of a single structural domain and adopt t...

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Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

et al. 2009

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Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin.

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Topological Switching between an α−β Parallel Protein and a Remarkably Helical Molten Globule

Cited by 14 publications

References 49 publications

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

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