Recombinant Flavobacterium johnsoniae Potassium-transporting ATPase C chain (kdpC)

How does kdpC functionally interact with other components of the KdpFABC complex?

KdpC functions as an essential component of the KdpFABC complex, which collectively mediates potassium transport across the bacterial membrane. Within this complex, kdpC works in conjunction with KdpF, KdpA, and KdpB subunits. Based on structural studies of KdpFABC complexes, kdpC remains relatively immobile during the transport cycle, contradicting earlier hypotheses about its potential gating function .

Current research indicates that rather than directly participating in channel formation, kdpC appears to stabilize the complex and potentially modulates the connections between the potassium binding sites in KdpA and the ATP hydrolysis activities in KdpB. The complex undergoes conformational changes following a Post-Albers cycle, including E1 and E2-P conformations, which have been partially characterized through techniques such as EPR and cryo-EM .

What expression systems are most effective for producing functional recombinant kdpC protein?

For successful expression of recombinant Flavobacterium johnsoniae kdpC, researchers should consider both prokaryotic and eukaryotic expression systems based on experimental requirements:

Prokaryotic systems:

• E. coli systems using vectors like pUC with ampicillin resistance markers have proven effective for initial cloning

• BL21(DE3) or Rosetta strains may improve expression of membrane proteins like kdpC

• Consider codon optimization for Flavobacterium johnsoniae genes when expressing in E. coli

Eukaryotic systems:

• HEK-293T cells have demonstrated effectiveness in expressing complex transmembrane proteins

• Vectors containing CMV promoter/enhancer elements support constitutive expression

• Adding N-terminal HA-tag and C-terminal c-Myc tags facilitates detection and purification

The choice between these systems should be guided by the intended application, with E. coli being more suitable for high-yield structural studies and eukaryotic systems potentially offering better post-translational modifications for functional studies.

What purification strategy ensures high purity and functional integrity of recombinant kdpC?

A comprehensive purification strategy for recombinant kdpC should include:

• Membrane fraction isolation:

  • Cell lysis using French press or sonication in buffer containing protease inhibitors

  • Differential centrifugation to isolate membrane fractions (30,000-100,000×g ultracentrifugation)

• Membrane protein solubilization:

  • Gentle detergents like n-dodecyl-β-D-maltoside (DDM) or CHAPS at concentrations above critical micelle concentration

  • Solubilization buffer containing stabilizing ions (particularly potassium)

• Affinity chromatography:

  • Utilizing the introduced tags (HA or c-Myc) for immunoaffinity purification

  • Metal affinity chromatography if His-tags are incorporated

• Quality assessment:

  • SDS-PAGE analysis coupled with western blotting

  • Mass spectrometry for sequence confirmation

  • Size-exclusion chromatography for oligomeric state determination

Storage in Tris-based buffer with 50% glycerol at -20°C maintains stability, though repeated freeze-thaw cycles should be avoided. For extended storage, aliquots should be kept at -80°C .

How should researchers design experiments to study kdpC function within the KdpFABC complex?

Designing robust experiments to study kdpC function requires integrating multiple approaches:

Structural analysis approaches:

• Cryo-EM studies to visualize different conformational states

• FRET or EPR spectroscopy to track conformational changes in the protein complex

• Molecular dynamics simulations to predict movements during transport cycle

Functional assays:

• ATPase activity measurements using colorimetric assays (e.g., malachite green)

• Potassium transport assays using radioactive isotopes (86Rb+ or 42K+)

• Membrane potential measurements using voltage-sensitive dyes

Interaction studies:

• Co-immunoprecipitation of kdpC with other Kdp subunits

• Crosslinking experiments followed by mass spectrometry

• Yeast two-hybrid or bacterial two-hybrid assays for protein-protein interactions

These approaches should be combined with site-directed mutagenesis to systematically probe structure-function relationships. Experimental controls should include kdpC deletion mutants and complementation studies to verify observed phenotypes.

What in silico approaches can improve experimental design for recombinant kdpC studies?

Computational approaches can significantly enhance experimental design for kdpC research:

Sequence analysis:

• Multiple sequence alignment to identify conserved residues across bacterial species

• Homology modeling based on related structures

• Prediction of transmembrane regions and potential interaction sites

Structure prediction and validation:

• Molecular modeling to predict kdpC tertiary structure

• Docking studies to analyze interactions with other Kdp subunits

• Energy minimization to identify stable conformations

Simulation methodologies:

• Molecular dynamics simulations in membrane environments

• Brownian dynamics to model potassium ion movement

• Quantum mechanics/molecular mechanics approaches for ATPase reaction mechanism

An effective integration of in silico and experimental approaches, as demonstrated in recent recombinant protein research, can significantly improve the production yield while minimizing costs and experimental iterations .

How can researchers reconcile contradictory findings about kdpC function in different experimental systems?

Contradictions in kdpC research findings can arise from multiple factors and require systematic resolution approaches:

Sources of contradictions:

• Different model organisms (e.g., E. coli vs. Flavobacterium johnsoniae)

• Varied experimental conditions affecting protein conformation

• Different recombinant constructs (tags, fusion proteins)

• Diverse measurement techniques with varying sensitivities

Resolution strategies:

• Direct comparison experiments:

  • Side-by-side testing under identical conditions

  • Use of multiple complementary techniques for each measurement

  • Statistical analysis of reproducibility

• Standardization approaches:

  • Development of reference materials and standard protocols

  • Detailed reporting of all experimental parameters

  • Open data sharing for independent verification

• Mechanistic investigations:

  • Detailed kinetic analyses to identify condition-dependent behaviors

  • Structure-function studies to identify contextual influences on protein behavior

For example, functional studies from Siebers and Altendorf reporting maximal phosphorylation of the KdpFABC complex contradicted other structural findings, likely due to different experimental conditions affecting conformational states .

What approaches can be used to study kdpC in the context of Flavobacterium johnsoniae physiology?

Investigating kdpC within the broader physiological context of Flavobacterium johnsoniae requires specialized approaches:

Genetic manipulation strategies:

• Creation of unmarked deletions using streptomycin-resistant rpsL mutants as background strains

• Complementation studies using pCP1-derived plasmids with copy numbers around 10 in F. johnsoniae

• Gene editing using CRISPR-Cas systems adapted for Flavobacterium

Physiological assessments:

• Growth kinetics under varying potassium concentrations

• Membrane potential measurements during potassium starvation/repletion

• Cell motility studies, as potassium transport may affect gliding motility in F. johnsoniae

Integration with other cellular systems:

• Potential interactions with the type IX secretion system (T9SS)

• Influence on cell surface adhesins like SprB and RemA

• Effects on environmental adaptation mechanisms

Studies should employ Flavobacterium johnsoniae ATCC 17061 strain UW101 as a reference wild-type, with appropriate antibiotic selection: ampicillin (100 μg/ml), cefoxitin (100 μg/ml), erythromycin (100 μg/ml), streptomycin (100 μg/ml), or tetracycline (20 μg/ml) .

What future directions are promising for recombinant kdpC research?

Future research on recombinant Flavobacterium johnsoniae kdpC should focus on several promising directions:

Structural biology advancements:

• Time-resolved cryo-EM to capture transient conformational states

• Neutron diffraction studies to precisely locate potassium ions

• Integration of AlphaFold-predicted structures with experimental validation

Biotechnological applications:

• Development of kdpC-based biosensors for potassium detection

• Engineering kdpC with modified ion selectivity for specialized applications

• Utilizing kdpC in nanodisc systems for membrane protein research platforms

System-level understanding:

• Multi-omics approaches to understand kdpC regulation in response to environmental changes

• Synthetic biology approaches to reconstruct minimal potassium transport systems

• Comparative studies across bacterial species to understand evolutionary adaptations

These approaches should employ a combination of computational modeling and experimental validation, as exemplified by recent work on biomimetic vector design containing recombinant proteins .