Cloning and characterization of the flavodoxin gene from Desulfovibrio desulfuricans

Helms, Larry R.; Swenson, Richard P.

doi:10.1016/0167-4781(91)90190-w

Cited by 29 publications

(19 citation statements)

References 6 publications

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“…Flavodoxins have been identified and isolated from several members of the Desulfovibrio genus [18,26,331. Most have been characterized in terms of molecular mass, amino acid composition and sequences, as well as visible spectroscopy (oxidized, one-electron-reduced and two-electron-reduced forms).…”

Section: Resultsmentioning

confidence: 99%

Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin

Caldeira

Palma

Regalla

et al. 1994

European Journal of Biochemistry

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Flavodoxin was isolated and purified from Desulfovibrio desulfidricans ATCC 27774, a sulfatereducing organism that can also utilize nitrate as an alternative electron acceptor. Mid-point oxidation-reduction potentials of this flavodoxin were determined by ultravioletlvisible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidizedsemiquinone forms at pH 6.7 and 25°C is -40 mV, while the value for the semiquinonehydroquinone forms (El), at the same pH, -387 mV. E2 varies linearly with pH, while E, is independent of pH at high values. However, at low pH (<7.0), this value is less negative, compatible with a redox-linked protonation of the flavodoxin hydroquinone. Flavodoxins are a group of small electron-transfer proteins [l-31 (= 15-23 kDa) isolated from different organisms, that contain a single FMN group bound non-covalently to the polypeptide chain. They can transfer two electrons at low and differentiated redox potentials.The way in which a flavodoxin's peptide chain modulates the redox properties of the FMN cofactor has been the major focus of the tridimensional structural studies. Thermodynamic data have been discussed in terms of the binding of the cofactor to the polypeptide chain in different redox states of the protein. The pH dependence of the mid-point oxidationreduction potentials has been extensively explored.To infer on factors controlling the electron-transfer process, X-ray diffraction and NMR spectroscopy have been valuable tools to reveal. the environment of the FMN group bound to the protein. Anacystis nidulans [9] and Chondrus crispus [lo, 111 flavodoxins from X-ray studies ; and structures were proposed from two-dimensional and three-dimensional NMR studies for Megasphaera elsdenii [12-151 and D. vulgaris [16, 171 In this study we present the isolation, purification and characterization of D. desulfuricans ATCC 27774 flavodoxin. Redox potentials were determined by potentiometric titrations coupled with visible and EPR techniques, at different pH values. The value of the dissociation constant of the FMN group was estimated by differential spectrophotometric titrations of FMN with apo-flavodoxin. The complete amino acid sequence of D. desulfuricans ATCC 27774 flavodoxin was obtained and its degree of similarity with other Desulfovibrio species flavodoxins is presented. A theoretical model of a tertiary structure obtained using the most similar sequence available (D. vulgaris flavodoxin) is predicted using molecular-modeling tools. D. vulgaris

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Section: Resultsmentioning

confidence: 99%

Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin

Caldeira

Palma

Regalla

et al. 1994

European Journal of Biochemistry

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“…Flavodoxin from D. desulfuricans (American Type Culture Collection strain 29577) was expressed in Escherichia coli and purified as described (39,40). In short, the apo-form of wild-type flavodoxin was isolated on a Q-Sepharose column and further purified by gel permeation on a Superdex-75 by using an FPLC system (Amersham-Amersham Pharmacia) (25).…”

Section: Methodsmentioning

confidence: 99%

Molecular crowding enhances native structure and stability of α/β protein flavodoxin

Stagg

Zhang²,

Cheung³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

256

276

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To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue singledomain ␣/␤ protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (⌬T m of 20°C at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20°C) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hardsphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central ␤-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.energy landscape theory ͉ excluded volume effect ͉ molecular simulations ͉ protein folding

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“…Recombinant D. desulfuricans [Essex 6] flavodoxin was expressed in E. coli transformed with the Bluescript phagmid containing the 1.8-kb TaqI insert, as previously described (Helms and Swenson, 1991;Helms, 1992). Uniformly 15N-enriched flavodoxin was prepared from M9 minimal medium with 15NH4C1 (Cambridge Isotopes, Andover, MA), supplied as the exclusive source of nitrogen.…”

Section: Protein Enrichment and Sample Preparationmentioning

confidence: 99%

“…Of the Desulfovibrio family, D. desulfuricans flavodoxin is the least homologous to D. vulgaris, having only a 47% residue correspondence, as shown in Fig. 1 (Helms and Swenson, 1991;Swenson and Krey, 1994). Resonance assignments for the wild-type protein also provide a reference point for examining the effects of single amino acid mutations on protein-cofactor interactions in this member of the Desulfovibrio family.…”

Section: Introductionmentioning

confidence: 95%

“…0925-2738/$ 6.00 + 1.00 9 1996 ESCOM Science Publishers B.V. I VYGS TTGN TEYTAE T I ARE LADAG I I I I I I I i i I I I I i I DD MSKVLIVFGS STGNTES IAQKLEELIAAGG 40 50 60 DV YEVD S RDAAS VEAGGL FEGFDLVL LGC S TW I I I I I I I I I I I I I I DD HEVT LLNAADAS AENLADGYDAVL FGCS AW 70 80 1992), two strains of D. gigas (ATCC 29494 and 19364) (Helms and Swenson, 1991), D. salexigens (ATCC 14822) (Helms et al, 1990), and D. vulgaris (Swenson and Krey, 1994) (Helms, 1992). Consequently, D. desulfuricans flavodoxin has the widest redox-potential span known for this family of proteins.…”

Section: Introductionmentioning

confidence: 98%

See 1 more Smart Citation

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Pollock¹,

Swenson

Stockman³

1996

J Biomol NMR

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Sequence-specific 1H and 15N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional 1H-15N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly 15N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping 1HN chemical shifts were resolved by a three-dimensional 1H-15N HMQC-NOESY-HMQC spectrum. Medium- and long-range NOEs, 3JNH alpha coupling constants, and 1HN exchange data indicate a secondary structure consisting of five parallel beta-strands and four alpha-helices with a topology similar to that of Desulfovibrio vulgaris [Hildenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal alpha-helices.

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Cloning and characterization of the flavodoxin gene from Desulfovibrio desulfuricans

Cited by 29 publications

References 6 publications

Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin

Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin

Molecular crowding enhances native structure and stability of α/β protein flavodoxin

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

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