Apparent local stability of the secondary structure of <i>Azotobacter vinelandii</i> holoflavodoxin II as probed by hydrogen exchange: Implications for redox potential regulation and flavodoxin folding

Steensma, E.; Nijman, Marcel J.; Bollen, Yves J.M.; Jager, P.A. de; Berg, W.A.M. van den; Dongen, W.M.A.M. van; Mierlo, C.P.M. van

doi:10.1002/pro.5560070210

Cited by 32 publications

(32 citation statements)

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“…The kinetic folding of apoflavodoxin (i.e. flavodoxin without the FMN cofactor) is described by the reaction, I off 7 U 7 I on 7 N, where U is unfolded protein and N is native apoflavodoxin, which is structurally identical to flavodoxin except for some dynamic disorder in the flavin-binding region (27,28). Apoflavodoxin folds autonomously, and the subsequent binding of FMN is the last step in flavodoxin folding (24).…”

mentioning

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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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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“…Given their key biological function and a series of practical facts (i.e. they were among the first proteins for which x-ray structures became available (3,4), their purification in the pre-recombinant era was relatively easy, and they are reasonably stable to handle), flavodoxins were soon found to be convenient models to investigate electron transfer and molecular recognition (1,2) and, more recently, protein stability (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and folding (18 -21). Based on molecular weight and sequence comparisons, they were divided in two families: short-chain and long-chain flavodoxins (the latter containing an extra ϳ20-residue segment, subsequently shown in Refs.…”

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confidence: 99%

The Long and Short Flavodoxins

López-Llano

Maldonado

Jain

et al. 2004

Journal of Biological Chemistry

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Flavodoxins are classified in two groups according to the presence or absence of a ϳ20-residue loop of unknown function. In the accompanying paper (36), we have shown that the differentiating loop from the longchain Anabaena PCC 7119 flavodoxin is a peripheral structural element that can be removed without preventing the proper folding of the apoprotein. Here we investigate the role played by the loop in the stability and folding mechanism of flavodoxin by comparing the equilibrium and kinetic behavior of the full-length protein with that of loop-lacking, shortened variants. We show that, when the loop is removed, the three-state equilibrium thermal unfolding of apoflavodoxin becomes two-state. Thus, the loop is responsible for the complexity shown by long-chain apoflavodoxins toward thermal denaturation. As for the folding reaction, both shortened and wild type apoflavodoxins display threestate behavior but their folding mechanisms clearly differ. Whereas the full-length protein populates an essentially off-pathway transient intermediate, the additional state observed in the folding of the shortened variant analyzed seems to be simply an alternative native conformation. This finding suggests that the long loop may also be responsible for the accumulation of the kinetic intermediate observed in the full-length protein. Most revealing, however, is that the influence of the loop on the overall conformational stability of apoflavodoxin is quite low and the natively folded shortened variant ⌬(120 -139) is almost as stable as the wild type protein.The fact that the loop, which is not required for a proper folding of the polypeptide, does not even play a significant role in increasing the conformational stability of the protein supports our proposal (36) that the differentiating loop of long-chain flavodoxins may be related to a recognition function, rather than serving a structural purpose.The flavodoxins are well known electron transfer proteins involved in both photosynthetic and non-photosynthetic reactions that carry a non-covalently bound FMN molecule as a redox center (1, 2). Given their key biological function and a series of practical facts (i.e. they were among the first proteins for which x-ray structures became available (3, 4), their purification in the pre-recombinant era was relatively easy, and they are reasonably stable to handle), flavodoxins were soon found to be convenient models to investigate electron transfer and molecular recognition (1, 2) and, more recently, protein stability (5-20) and folding (18 -21). Based on molecular weight and sequence comparisons, they were divided in two families: short-chain and long-chain flavodoxins (the latter containing an extra ϳ20-residue segment, subsequently shown in Refs. 22 and 23 to form a loop in the folded protein, as highlighted in Fig. 1 of our accompanying article (36)). Despite the wealth of structural and functional information available for several flavodoxins of either family, it is still unclear whether the differentiating loop plays a structura...

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“…The flavodoxin-like topology is considered to be one the most ancient protein folds (60). While the flavodoxin protein itself is absent from higher kingdoms of life such as plants and animals (61), it is present in bacteria and many algal species (62). Furthermore, flavodoxin-like folds are found as domains in many crucial multi-domain eukaryotic proteins such as cytochrome P450 reductase (63) and nitrogen oxide synthase (64).…”

Section: Elucidating Cotranslational Folding Of Flavodoxinmentioning

confidence: 99%

“…Polarized time-resolved fluorescence data are related to steady-state fluorescence anisotropy according to the following relation (62):…”

Section: Eq 13mentioning

confidence: 99%

“…After binding FMN with nanomolar affinity, the protein relaxes to an energetically even more favourable state with picomolar binding affinity (74). As a result, many amino acid residues dispersed throughout the structure of flavodoxin become stabilized against unfolding (62,74). The stability of flavodoxin is so high that FMN needs to be released first before global unfolding of the protein can occur (65).…”

Section: Exploration Of the Folding Of Flavodoxin From A Vinelandiimentioning

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: Implications for redox potential regulation and flavodoxin folding

Cited by 32 publications

References 65 publications

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

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

The Long and Short Flavodoxins

The ribosome regulates flavodoxin folding

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