Predissociated Dimers and Molten Globule Monomers in the Equilibrium Unfolding of Yeast Glutathione Reductase

Louzada, Paulo Roberto; Sebollela, Adriano; Scaramello, Marcelo E.; Ferreira, Sérgio T.

doi:10.1016/s0006-3495(03)74743-2

Cited by 25 publications

(15 citation statements)

References 36 publications

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“…However, this result is consistent with that of several other dimeric proteins including factor for inversion stimulation protein, 50 the ATPase SecA, 72 organophosphorus hydrolase, 73 triosephosphate isomerase 74,75 and glutathione reductase 76 where a dimeric intermediate has been observed. Like apo-S100B, each of these proteins has the appearance of a transition corresponding to a F 2 ⇄ I 2 step at relatively low GuHCl concentrations (b 2 M), indicative that the dissociation of the dimer is a less favourable process.…”

Section: Discussionsupporting

confidence: 87%

Identification of a Dimeric Intermediate in the Unfolding Pathway for the Calcium-Binding Protein S100B

Shaw

Marlatt

Ferguson

et al. 2008

Journal of Molecular Biology

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Section: Discussionsupporting

confidence: 87%

Identification of a Dimeric Intermediate in the Unfolding Pathway for the Calcium-Binding Protein S100B

Shaw

Marlatt

Ferguson

et al. 2008

Journal of Molecular Biology

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“…Although A300 is conserved in most enzymes, its replacement with Thr, as (34). However, after reactivation with cysteine and FAD, the variant recovered some activity, suggesting that the N302S variant may be in a partially folded and expanded dimeric state, like unfolding intermediates previously described for other flavoproteins (6,23). This partially unfolded form may partly recover the compact dimer structure after reactivation.…”

Section: Discussionmentioning

confidence: 74%

Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

2011

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Water-forming NADH oxidases (encoded by noxE, nox2, or nox) are flavoproteins generally implicated in the aerobic survival of microaerophilic bacteria, such as lactic acid bacteria. However, some natural Lactococcus lactis strains produce an inactive NoxE. We examined the role of NoxE in the oxygen tolerance of L. lactis in the rich synthetic medium GM17. Inactivation of noxE suppressed 95% of NADH oxidase activity but only slightly affected aerobic growth, oxidative stress resistance, and NAD regeneration. However, noxE inactivation strongly impaired oxygen consumption and mixed-acid fermentation. We found that the A303T mutation is responsible for the loss of activity of a naturally occurring variant of NoxE. Replacement of A303 with T or G or of G307 with S or A by site-directed mutagenesis led to NoxE aggregation and the total loss of activity. We demonstrated that L299 is involved in NoxE activity, probably contributing to positioning flavin adenine dinucleotide (FAD) in the active site. These residues are part of the strongly conserved sequence LA(T)XXA XXXG included in an alpha helix that is present in other flavoprotein disulfide reductase (FDR) family flavoproteins that display very similar three-dimensional structures.Water-forming NADH oxidases (Nox, NoxE, or Nox2) are flavoproteins involved in the aerobic growth and survival of lactic acid bacteria (LAB) under fermentation conditions (7). Their inactivation in several streptococci strongly reduces the aerobic growth. Inactivation of nox2 in Streptococcus pneumoniae and Streptococcus agalactiae reduces growth in aerated media by 80% (37, 38). The growth defect of the S. agalactiae mutant has been attributed to a defect in fatty acid production resulting from nonproduction of the fatty acid precursor acetyl-coenzyme A (CoA) due to strictly homolactic fermentation (37). Nox2 inactivation in Streptococcus mutans also leads to strict homolactic fermentation on glucose; however, it does not affect growth on glucose but greatly reduces growth on mannitol (11). In Streptococcus pyogenes, Nox2 inactivation completely abolishes aerobic growth under carbon limitation conditions and increases sensitivity to the superoxide-generating agent paraquat (methyl viologen). This growth defect has been attributed to hydrogen peroxide (H 2 O 2 ) accumulation (10). NoxE is the principal enzyme displaying NADH oxidase (NOX) activity in Lactococcus lactis (95% of the NADH oxidase activity of L. lactis TIL46), a LAB widely used in industrial milk fermentations and generally considered a model LAB. However, NoxE inactivation in L. lactis reduces the growth rate in milk by only 20%, although it strongly impairs oxygen consumption (34). Moreover, we isolated two natural L. lactis strains without detectable NADH oxidase activity from matured cheese (34), suggesting that NoxE is not important for L. lactis survival in dairy media. In one natural NADH oxidase-negative strain, the noxE gene was complete but the enzyme appeared to be inactive. Several water-forming NADH oxidases from...

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“…Recently, Louzada et al . (36) proposed the mechanism of a quaternary structure (dimer) formation in the folding pathway of glutathione reductase (GR). They proposed that the assembly of the native GR dimer proceed via the initial formation of molten-globule monomers that dimerize to form a partially folded, expanded dimer.…”

Section: Discussionmentioning

confidence: 99%

Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone

Ahn

Choi²,

Kim³

et al. 2005

Nucleic Acids Research

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Contrary to the results of direct expression, various human proteins (ferritin light-chain, epithermal growth factor, interleukin-2, prepro-ghrelin, deletion mutants of glutamate decarboxylase and arginine deiminase, and mini-proinsulin) were all soluble in Escherichia coli cytoplasm when expressed with the N-terminus fusion of ferritin heavy-chain (FTN-H). Through systematic investigations, we have found that a specific peptide motif within FTN-H has a high affinity to HSP70 chaperone DnaK, and that the peptide motif was composed of a hydrophobic core of three residues (Ile, Phe and Leu) and two flanking regions enriched with polar residues (Gly, Gln and Arg). It was also observed that all the recombinant proteins expressed with the fusion of FTN-H formed spherical nanoparticles with diameters of 10–15 nm, as confirmed by the transmission electron microscopy image. The protein nanoparticles are non-covalently cross-linked supra-molecules formed by the self-assembly function of FTN-H. Upon the formation of the supra-molecule, its size is likely to be limited by the assembly properties of FTN-H, thereby keeping the self-assembled particles soluble. This study reports on the dual function of FTN-H for fusion expression and solubility enhancement of heterologous proteins: (i) high-affinity interaction with DnaK and (ii) formation of self-assembled supra-molecules with limited and constant sizes, thereby avoiding the undesirable formation of insoluble macro-aggregates of heterologous proteins.

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Predissociated Dimers and Molten Globule Monomers in the Equilibrium Unfolding of Yeast Glutathione Reductase

Cited by 25 publications

References 36 publications

Identification of a Dimeric Intermediate in the Unfolding Pathway for the Calcium-Binding Protein S100B

Identification of a Dimeric Intermediate in the Unfolding Pathway for the Calcium-Binding Protein S100B

Identification of a Conserved Sequence in Flavoproteins Essential for the Correct Conformation and Activity of the NADH Oxidase NoxE of Lactococcus lactis

Heterologous gene expression using self-assembled supra-molecules with high affinity for HSP70 chaperone

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