Apoflavodoxin: Structure, stability, and FMN binding

Maldonado, Susana; Lostao, Anabel; Irún, María Pilar; Fernández‐Recio, Juan; Genzor, Carlos G.; Gonzalez, Elena; Rubio, José Antonio; Luquita, Alejandra; Daoudi, Fatna; Sancho, Javier

doi:10.1016/s0300-9084(00)88876-8

Cited by 17 publications

(18 citation statements)

References 19 publications

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“…At this point, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 1mM and the cells were allowed to grow for at least 3 h and then pelleted and washed once with 0.15 M NaCl. After centrifugation, the cells were dissolved in 50 mM Tris-HCl buffer, pH 8, In addition, the Anabaena flavodoxin W57A mutant, where the highly conserved tryptophan residue of the FMN binding site is substituted by alanine, was expressed and purified as described [30].…”

Section: Experimental Dna Isolation and Pcr Amplification Of The Fldmentioning

confidence: 99%

“…Our laboratory has used over the years the flavodoxin from a different specie (Anabaena PCC7119) as a model protein to investigate protein stability, protein folding and binding [29][30][31][32][33][34][35]. From the structural point of view, flavodoxins are α/β proteins and bind, at the C-terminal end of the β-sheet, a molecule of flavin mononucleotide (FMN) that confers redox properties to the protein [36].…”

Section: Introductionmentioning

confidence: 99%

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Towards a new therapeutic target: Helicobacter pylori flavodoxin

Cremades

Bueno

Toja³

et al. 2005

Biophysical Chemistry

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Section: Experimental Dna Isolation and Pcr Amplification Of The Fldmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Towards a new therapeutic target: Helicobacter pylori flavodoxin

Cremades

Bueno

Toja³

et al. 2005

Biophysical Chemistry

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“…5A). For this mechanism, the equations of the relaxation rates ( 1 and 2 ) and reduced amplitudes (normalized between 0 and 1 as described previously (21) vodoxin Stability and in the Stability of the Equilibrium Intermediate-The conformational stability of the apoflavodoxins of several species has been investigated, and it seems to be generally low (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). As for the stability of the holoform (carrying the FMN cofactor), there have been divergent reports pointing to either a significant or a rather marginal stabilization imparted by the presence of the prosthetic group (13,18).…”

Section: Kinetics Of Unfolding and Refolding Of ⌬(120 -139)-shortenedmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…The conformational stability and/or folding kinetics of three short apoflavodoxins (two different strains of Desulfovibrio desulfuricans (13,20) and Desulfovibrio vulgaris (18)) and two long ones (Azotobacter vinelandii (14 -17) and Anabaena PCC 7119 (5)(6)(7)(8)(9)(10)(11)(12)21)) have been reported. The available data, which have not always been gathered under the same conditions for the different proteins, suggest (but do not prove) that the long flavodoxins could display a more complex equilibrium and folding behavior than the short ones.…”

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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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Native‐specific stabilization of flavodoxin by the FMN cofactor: Structural and thermodynamical explanation

Campos

Sancho

2006

Proteins

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Flavodoxins are useful models to investigate protein/cofactor interactions. The binding energy of the apoflavodoxin-FMN complex is high and therefore the holoflavodoxin is expected to be more stable than the apoprotein. This expectation has been challenged by reports on the stability of Desulfovibrio desulfuricans flavodoxin indicating that FMN binds to the unfolded polypeptide with similar affinity as to the native state, thus causing no net effect on protein stability. In previous work, we have analyzed in detail the stability of the apoflavodoxin from Anabaena PCC 7119 and the energetics of its functional complex with FMN. Here, we use the Anabaena holoprotein to directly investigate the contribution of the bound cofactor to protein stability through a detailed analysis of the chemical and thermal denaturation equilibria. Our data clearly shows that FMN binding largely stabilizes the protein towards both chemical and thermal denaturation, and that the stabilization observed at 25 degrees C in low ionic strength conditions is precisely the one expected if full release of the cofactor takes place upon flavodoxin unfolding. On the other hand, the binding of FMN to the native polypeptide is shown to simplify the thermal unfolding so that, while apoflavodoxin follows a three-state mechanism, the holoprotein unfolds in a two-state fashion. Comparison of the X-ray structure of native apoflavodoxin with the phi-structure of the thermal intermediate indicates that the increase in cooperativity driven by the cofactor originates in its preferential binding to the native state, which is a consequence of the disorganization in the intermediate of the FMN binding loops and of an adjacent longer loop.

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Apoflavodoxin: Structure, stability, and FMN binding

Cited by 17 publications

References 19 publications

Towards a new therapeutic target: Helicobacter pylori flavodoxin

Towards a new therapeutic target: Helicobacter pylori flavodoxin

The Long and Short Flavodoxins

Native‐specific stabilization of flavodoxin by the FMN cofactor: Structural and thermodynamical explanation

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