Recombinant Desulfovibrio vulgaris Flavodoxin (DvMF_1143)

What is Desulfovibrio vulgaris Flavodoxin (DvMF_1143) and what are its fundamental properties?

Desulfovibrio vulgaris Flavodoxin (DvMF_1143) is an electron transfer protein from the anaerobic bacterium Desulfovibrio vulgaris (Hildenborough). It contains flavin mononucleotide (FMN) as its prosthetic group and functions as an electron shuttle in various redox-based pathways. The recombinant protein, when expressed in E. coli, is identical to the wild-type protein except for the absence of the N-terminal methionine residue . As a member of the flavodoxin family, it belongs to a class of small electron transfer proteins widely present in bacteria and absent in vertebrates .

How does the structure of DvMF_1143 relate to its function?

The structural features of DvMF_1143, particularly the FMN binding site, directly influence its electron transfer capabilities. Like other flavodoxins, DvMF_1143 likely has conserved tyrosine residues that flank the flavin on both sides, contributing to its redox properties . The protein structure includes dynamic surface loops that can adopt different conformations when binding to partner proteins, facilitating efficient electron transfer . These structural characteristics allow flavodoxins to participate in various metabolic pathways, serving as versatile electron carriers in bacterial systems .

What is the recommended method for recombinant expression of DvMF_1143?

For high-level expression of DvMF_1143, the gene should be subcloned as a minimal insert behind the tac promoter of plasmid pDK6 in Escherichia coli. This approach has been demonstrated to yield 3-4% of total soluble protein . The recombinant protein lacks the N-terminal methionine compared to the wild-type but maintains identical properties otherwise . For optimal results, expression conditions should be carefully controlled with respect to temperature, induction timing, and media composition to ensure proper folding and FMN incorporation.

How can researchers optimize the purification of recombinant DvMF_1143?

Methodology for DvMF_1143 purification should consider its biochemical properties. Based on studies with similar flavodoxins, a multi-step purification protocol is recommended:

• Cell lysis: Use sonication or pressure-based disruption in buffer containing protease inhibitors

• Initial clarification: Centrifugation at 20,000g followed by ammonium sulfate fractionation

• Chromatographic separation: Ion exchange chromatography (e.g., DEAE) followed by gel filtration

• Quality assessment: Purity can be verified through SDS-PAGE and spectroscopic methods that monitor the characteristic absorption spectrum of bound FMN

The distinctive yellow color of flavodoxins due to bound FMN provides a visual indicator during purification .

What are the redox potentials of DvMF_1143 and how are they measured?

DvMF_1143 exhibits two distinct redox couples with the following potentials at pH 7.0 and 25°C:

• Oxidized flavodoxin/flavodoxin semiquinone (E2): -143 mV

• Flavodoxin semiquinone/flavodoxin hydroquinone (E1): -440 mV

E2 varies linearly with pH (slope = -59 mV), while E1 is pH-independent at high pH values but becomes less negative with decreasing pH below 7.5, indicating redox-linked protonation of the flavodoxin hydroquinone .

These potentials can be measured using spectroelectrochemical techniques where spectral changes in the protein are monitored during potentiometric titration using suitable mediators. The formation of the semiquinone state is typically confirmed by the development of a blue color .

How do charge-charge interactions influence the redox properties of DvMF_1143?

Advanced research has shown that charge-charge interactions between different parts of the flavin hydroquinone play a crucial role in determining the E1 redox potential in flavodoxins . The protein environment around the FMN modifies its intrinsic properties, creating the characteristic low redox potentials that make flavodoxins suitable for specific electron transfer reactions .

To investigate these interactions, researchers can employ site-directed mutagenesis to alter charged residues near the FMN binding site, followed by redox potential measurements to assess the impact of these modifications. Computational methods such as molecular dynamics simulations and quantum mechanical calculations can further elucidate the electrostatic environment surrounding the flavin.

What techniques can be used to study electron transfer mechanisms involving DvMF_1143?

Several advanced biophysical methods can be employed to investigate electron transfer mechanisms:

• Laser flash photolysis: Monitors transient species formed during electron transfer reactions

• Stopped-flow spectroscopy: Measures reaction kinetics on millisecond timescales

• NMR chemical shift mapping: Identifies interaction surfaces between DvMF_1143 and its partners

• Protein-protein docking simulations: Predicts binding orientations that facilitate electron transfer

Studies with other flavodoxins have shown that they form mutually exclusive complexes with their electron-donating and electron-accepting partners, requiring conformational changes for interconversion .

How can researchers investigate the dynamics of DvMF_1143 using NMR spectroscopy?

NMR spectroscopy offers powerful approaches to study flavodoxin dynamics:

• 19F-NMR with fluorine-labeled tyrosines: This technique can reveal different environments affecting conserved tyrosines flanking the flavin and identify dynamic regions potentially involved in partner binding. Particularly useful is measuring the 19F signals of tyrosines adjacent to the flavin (such as those equivalent to Y53 and Y90 in other flavodoxins), which are paramagnetically relaxed when the protein adopts its semiquinone state .

• Temperature-dependent studies: Analyzing chemical shifts and linewidths at various temperatures can reveal dynamics affecting loops close to the flavin and regions that bind to partners .

• 15N relaxation analysis: While focused on rapid motions in the ps-ns timescale, this approach can identify surface loops with lower backbone order parameters than the protein core .

What are the known physiological partners of flavodoxins and how can these interactions be characterized?

While specific partners of DvMF_1143 aren't directly identified in the available literature, flavodoxins generally interact with:

• NADPH:ferredoxin oxidoreductase as an electron acceptor

• Various enzymes as an electron donor, including:

  • Ribonucleotide reductase

  • Biotin synthase

  • Pyruvate formate lyase

  • Cobalamin-dependent methionine synthase

These interactions can be characterized through:

• NMR chemical shift mapping to identify binding surfaces

• Co-crystallization or cryo-electron microscopy to determine complex structures

• Isothermal titration calorimetry to measure binding thermodynamics

• Surface plasmon resonance to evaluate binding kinetics

Research indicates that flavodoxins bind to unique overlapping sites on their physiological partners, precluding the formation of ternary complexes .

How does DvMF_1143 compare with flavodoxins from other bacterial species?

Comparative analysis of flavodoxins from different bacterial species reveals both conserved features and species-specific adaptations:

Flavodoxins from pathogenic bacteria like Helicobacter pylori and Clostridioides difficile have gained particular attention for their potential as therapeutic targets .

What are the potential applications of DvMF_1143 in biotechnology research?

DvMF_1143 has several potential research applications:

• Biocatalysis: As an electron carrier in redox reactions for biosynthesis of valuable compounds

• Biosensors: Development of electrochemical or optical sensors for monitoring redox processes

• Antimicrobial research: Model system for studying flavodoxin-based drug targeting against pathogenic bacteria, as flavodoxins are:

  • Essential for survival in some bacterial pathogens

  • Absent in vertebrates

  • Potential therapeutic targets

• Structural biology reference: Well-characterized model system for studying protein-FMN interactions and electron transfer mechanisms

• Agricultural biotechnology: Understanding flavodoxin functions could inform transgenic expression strategies in plants, where introduced flavodoxins have been shown to increase tolerance to multiple stresses and iron deficit

How can researchers utilize DvMF_1143 to study the role of flavodoxins in stress responses?

DvMF_1143 can serve as a model system to investigate stress response mechanisms involving flavodoxins:

• Iron limitation studies: Compare the function of DvMF_1143 with ferredoxins under varying iron concentrations to understand how flavodoxins replace ferredoxins during iron deficiency. Bacterial species like Clostridioides difficile show increased expression of specific flavodoxins (e.g., fldX) with decreasing iron concentration .

• Oxidative stress responses: Examine how DvMF_1143 maintains electron flow under oxidative conditions. In C. difficile, flavodoxin fldX shows increased expression under different oxidative stress conditions at both RNA and protein levels .

• Transgenic expression studies: Compare the properties of DvMF_1143 with flavodoxins expressed in transgenic plants, which exhibit enhanced tolerance to multiple stresses through mechanisms similar to those in microorganisms .

Methodology should include gene expression analysis under different stress conditions, protein activity assays, and interaction studies with stress-related pathways.

How can X-ray crystallography and cryo-electron microscopy complement NMR studies of DvMF_1143?

A multi-technique structural biology approach provides comprehensive insights:

• X-ray crystallography: Delivers high-resolution static structures revealing the precise arrangement of the FMN binding pocket and key residues involved in electron transfer. Crystallographic data obtained at different temperatures can identify regions with higher mobility .

• Cryo-electron microscopy: Particularly valuable for studying DvMF_1143 in complex with larger partner proteins where crystallization may be challenging.

• NMR spectroscopy: Provides dynamic information complementing the static crystal structures, revealing that loops surrounding the flavin access diverse conformations relevant to adaptive binding .

Together, these techniques can map conformational states throughout the catalytic cycle, connecting structure to function. For instance, crystallography might capture the ground state, while NMR can identify transient states during partner binding and electron transfer.

What computational methods are valuable for studying DvMF_1143's function and interactions?

Advanced computational approaches offer insights beyond experimental limitations:

• Molecular dynamics simulations: Can model the dynamic behavior of DvMF_1143, particularly the motions of surface loops that may participate in binding to partners. These simulations can investigate how the protein responds to changes in redox state.

• Quantum mechanics/molecular mechanics (QM/MM): Essential for modeling the electronic properties of the FMN cofactor and its interactions with the protein environment, particularly for understanding the unusual redox potentials of flavodoxins.

• Protein-protein docking: Predicts binding modes between DvMF_1143 and its electron transfer partners, generating testable hypotheses about interfacial residues.

• Sequence conservation analysis: Examining >500 different flavodoxin sequences can identify critical residues for partner binding versus regions displaying amino acid type conservation without retention of specific residues .

These computational tools should be integrated with experimental validation for maximum impact on understanding DvMF_1143 function.