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

Nabuurs, Sanne M.; Kort, Bregje J. de; Westphal, Adrie H.; Mierlo, C.P.M. van

doi:10.1007/s00249-009-0556-4

Cited by 17 publications

(23 citation statements)

References 46 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: 89%

“…As FMN synthesis is relatively slow, it takes much longer to assemble all the protein in holo form than the time in which the maximum of apo-protein is synthesized [57]. Due to amino acid residue replacement, F44Y, W128F, W167F or W128F/W167F apoflavodoxin variants are destabilized compared to wild-type protein [38,45,51,52]. Thus, these apo-proteins are more efficiently degraded by intracellular proteases before they can take advantage of the stabilization conferred through binding of FMN.…”

Section: Discussionmentioning

confidence: 96%

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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: 89%

Section: Discussionmentioning

confidence: 96%

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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“…23 Longrange interactions in unfolded apoflavodoxin studied using site-directed spin-labeling and paramagnetic relaxation enhancement showed that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin. 24 Energetics and position specificity of non-native hydrophobic interactions studied by a continuum coarsegrained chain model in the Fyn SH3 domain predicted that energetically significant non-native interactions led to acceleration or deceleration of the folding rate and energetic importance of the predicted non-native interactions confirmed experimentally conclude that specific non-native interactions can significantly influence folding energetics. 25 Gromiha and Selvaraj 3 proposed the novel parameter long-range order (LRO) based on the number of longrange contacts present in the 3D structures to predict folding rates of two-state proteins.…”

Section: Introductionmentioning

confidence: 71%

Application of long‐range order to predict unfolding rates of two‐state proteins

Balasubramanian

Selvaraj

2010

Proteins

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Predicting the experimental unfolding rates of two-state proteins and models describing the unfolding rates of these proteins is quite limited because of the complexity present in the unfolding mechanism and the lack of experimental unfolding data compared with folding data. In this work, 25 two-state proteins characterized by Maxwell et al. (Protein Sci 2005;14:602–616) using a consensus set of experimental conditions were taken, and the parameter long-range order (LRO) derived from their three-dimensional structures were related with their experimental unfolding rates ln(k(u)). From the total data set of 30 proteins used by Maxwell et al. (Protein Sci 2005;14:602–616), five slow-unfolding proteins with very low unfolding rates were considered to be outliers and were not included in our data set. Except all beta structural class, LRO of both the all-alpha and mixed-class proteins showed a strong inverse correlation of r = -0.99 and -0.88, respectively, with experimental ln(k(u)). LRO shows a correlation of -0.62 with experimental ln(k(u)) for all-beta proteins. For predicting the unfolding rates, a simple statistical method has been used and linear regression equations were developed for individual structural classes of proteins using LRO, and the results obtained showed a better agreement with experimental results.

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“…Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. This ordered core is gradually extended upon decreasing denaturant concentration [34], [35], [36], [37]. NMR spectroscopy detects formation of apoflavodoxin’s molten globule in an indirect manner through disappearance of resonances of unfolded protein.…”

Section: Discussionmentioning

confidence: 99%

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

et al. 2012

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Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-β parallel protein.

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

Cited by 17 publications

References 46 publications

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

Application of long‐range order to predict unfolding rates of two‐state proteins

Illuminating the Off-Pathway Nature of the Molten Globule Folding Intermediate of an α-β Parallel Protein

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