Redox Properties of Wild‐Type, Cys69Ala, and Cys69Ser <i>Azotobacter Vinelandii</i> Flavodoxin II as Measured by Cyclic Voltammetry and EPR Spectroscopy

Steensma, E.; Heering, Hendrik A.; Hagen, Wilfred R.; Mierlo, C.P.M. van

doi:10.1111/j.1432-1033.1996.00167.x

Cited by 37 publications

(41 citation statements)

References 34 publications

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“…In all experiments except for the ones utilizing dye-labeled protein molecules, the C69A variant of apoflavodoxin was used, in which the single cysteine residue 69 is replaced by an alanine residue to avoid covalent dimerization of apoflavodoxin. This protein variant is similar to wild-type apoflavodoxin regarding both redox potential of holoprotein and stability of apoprotein (25,41).…”

Section: Methodsmentioning

confidence: 78%

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Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding

Engel

Westphal

Huberts

et al. 2008

Journal of Biological Chemistry

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To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37 ؎ 3 Å at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol ؊1 . The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9°C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.

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Section: Methodsmentioning

confidence: 78%

“…This nonproductive aggregation can compete with productive folding. However, flavodoxin is overexpressed in the cytoplasm of E. coli and folds to its native functional form at high yield without noticeable problems (41).…”

Section: Discussionmentioning

confidence: 99%

Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding

Engel

Westphal

Huberts

et al. 2008

Journal of Biological Chemistry

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“…This protein variant is largely similar to wild-type flavodoxin regarding both redox potential of holoprotein and stability of apoprotein (37,38). Subsequently, in this protein variant, the phenylalanine at position 44 was substituted for a tyrosine using site-directed mutagenesis.…”

Section: Methodsmentioning

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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“…These variants are shortened at the C-terminus by five or ten amino acids, respectively. To avoid covalent protein dimerization, the single cysteine at position 69 of flavodoxin was replaced by an alanine in all constructs [44]. Plasmids were transformed in E. coli strain BL21(DE3) Δtig::kan for expression of RNCs.…”

Section: Engineering Rncsmentioning

confidence: 99%

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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Redox Properties of Wild‐Type, Cys69Ala, and Cys69Ser Azotobacter Vinelandii Flavodoxin II as Measured by Cyclic Voltammetry and EPR Spectroscopy

Cited by 37 publications

References 34 publications

Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding

Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding

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

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

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