Recombinant Thermobifida fusca NADH-quinone oxidoreductase subunit K (nuoK)

What is Thermobifida fusca NADH-quinone oxidoreductase subunit K and what is its significance in research?

Thermobifida fusca NADH-quinone oxidoreductase subunit K (nuoK) is a membrane protein subunit of respiratory complex I (NADH:ubiquinone oxidoreductase, EC 1.6.99.5) from the thermophilic soil bacterium Thermobifida fusca. This protein consists of 96 amino acids and is encoded by the nuoK gene (locus tag Tfu_2685) . As part of the membrane domain of complex I, it likely participates in proton translocation across the bacterial membrane during respiration.

The significance of nuoK in research stems from several factors:

• It represents a component of the electron transport chain in a thermophilic organism

• It serves as a model for understanding respiratory complex assembly and function

• Its thermostability offers insights into protein adaptations to high temperatures

• It provides opportunities to study membrane protein structure-function relationships

What is known about Thermobifida fusca as a model organism?

Thermobifida fusca is a moderately thermophilic, aerobic, filamentous soil bacterium belonging to the phylum Actinobacteria . It has gained attention in research for several reasons:

• It produces an array of thermostable plant cell wall hydrolytic enzymes, making it biotechnologically valuable

• It can degrade most major plant cell wall polymers except lignin and pectin

• It possesses a single circular chromosome of 3,642,249 base pairs with 3,117 predicted coding sequences

• It grows optimally at elevated temperatures (around 50°C)

• Its genome has been fully sequenced and analyzed (GenBank accession: CP000088)

T. fusca was first isolated from decaying wood and has been primarily studied for its cellulolytic capabilities, though its bioenergetic systems including respiratory complexes remain less explored .

What is the amino acid sequence and basic properties of Thermobifida fusca nuoK?

The complete amino acid sequence of T. fusca nuoK (UniProt ID: Q47LF4) is:

MNYIVLAAIVFTIGAVGVLVRRNAIIVFMCVELMLNACNLAFVAFARMHGGIEGQVIAFFVMVVAAAEVVVGLAIIMQIFRTRRSASIDDANLLKN

Key properties of this protein include:

• Length: 96 amino acids

• Molecular weight: Approximately 10-11 kDa

• Highly hydrophobic nature, consistent with its role as a membrane protein

• Contains multiple predicted transmembrane helices

• Can be produced recombinantly with N-terminal His-tag for purification purposes

• When expressed recombinantly in E. coli, maintains its structural integrity

What expression systems are optimal for recombinant production of Thermobifida fusca nuoK?

Based on available research protocols, the optimal expression system for recombinant T. fusca nuoK involves:

• Host organism: Escherichia coli is the preferred heterologous expression host

• Expression vector: Plasmids containing an N-terminal His-tag for affinity purification

• Strain selection: E. coli strains optimized for membrane protein expression (such as C41/C43 or Lemo21) may improve yields

• Growth conditions:

  • Medium: Standard LB or 2×YT medium with appropriate antibiotics

  • Temperature: Initial growth at 37°C until mid-log phase, followed by temperature reduction to 25-30°C post-induction

  • Induction: IPTG at concentrations of 0.1-0.5 mM

  • Expression time: 4-6 hours post-induction or overnight at lower temperatures

Since nuoK is a membrane protein, expression optimization should focus on reducing toxicity while maintaining proper membrane insertion and folding.

What purification strategies yield highest purity and activity for recombinant nuoK?

Based on commercial protein preparation methods and standard membrane protein protocols, an effective purification strategy for His-tagged T. fusca nuoK includes:

• Cell lysis:

  • Mechanical disruption (sonication, French press, or high-pressure homogenization)

  • Buffer composition: Typically Tris or phosphate-based buffer (pH 7.5-8.0) containing 150-300 mM NaCl

• Membrane fraction isolation:

  • Differential centrifugation to separate membrane fraction (typically 100,000×g ultracentrifugation)

  • Membrane solubilization using appropriate detergents (e.g., n-dodecyl-β-D-maltoside, CHAPS, or digitonin)

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Washing with increasing imidazole concentrations (10-50 mM)

  • Elution with higher imidazole concentrations (250-500 mM)

• Further purification:

  • Size exclusion chromatography to remove aggregates and assess oligomeric state

  • Ion exchange chromatography for removal of remaining contaminants

The final purified protein typically shows >90% purity as determined by SDS-PAGE .

What are the optimal storage conditions for maintaining stability of purified nuoK?

For optimal stability of purified recombinant T. fusca nuoK, the following storage conditions are recommended:

• Short-term storage (up to 1 week):

  • Temperature: 4°C

  • Buffer: Tris/PBS-based buffer containing appropriate detergent above critical micelle concentration

• Long-term storage:

  • Temperature: -20°C or preferably -80°C

  • Buffer additives: 6% trehalose as a cryoprotectant

  • Format: Aliquoted samples to avoid repeated freeze-thaw cycles

• Lyophilization considerations:

  • Lyophilized powder format provides extended stability

  • Upon receipt, brief centrifugation is recommended before opening the vial

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Critical precautions:

  • Avoid repeated freeze-thaw cycles as they significantly reduce protein stability

  • For working aliquots, storage at 4°C is recommended for up to one week

What biophysical techniques are most informative for studying nuoK structure and function?

For comprehensive structural and functional characterization of T. fusca nuoK, researchers should consider the following biophysical techniques:

• Membrane protein structure determination:

  • Cryo-electron microscopy (cryo-EM): Particularly valuable for membrane protein complexes

  • X-ray crystallography: If well-diffracting crystals can be obtained

  • NMR spectroscopy: For dynamics studies of specifically labeled proteins

• Secondary structure analysis:

  • Circular dichroism (CD) spectroscopy: To determine secondary structure content and thermal stability

  • Fourier-transform infrared spectroscopy (FTIR): Particularly valuable for membrane proteins

• Protein-protein interactions:

  • Crosslinking coupled with mass spectrometry: To identify interaction interfaces

  • Blue native PAGE: To assess complex formation and stability

  • Microscale thermophoresis: For quantitative binding studies

• Functional assays:

  • Proteoliposome reconstitution: To measure proton pumping activity

  • NADH:ubiquinone oxidoreductase activity assays: When incorporated into intact complex

  • Membrane potential measurements: Using fluorescent probes in reconstituted systems

• Thermal stability assessment:

  • Differential scanning calorimetry: To determine melting temperatures

  • Thermal shift assays: For high-throughput stability screening

How do researchers investigate the role of nuoK in proton translocation?

Investigating the role of T. fusca nuoK in proton translocation requires specialized approaches:

• Site-directed mutagenesis studies:

  • Identification of conserved charged or polar residues within transmembrane domains

  • Systematic mutation of candidate proton-carrying residues

  • Functional characterization of mutants in reconstituted systems

• Proton translocation assays:

  • Reconstitution into proteoliposomes with pH-sensitive fluorescent dyes

  • pH electrode-based measurements of proton pumping

  • Membrane potential measurements using voltage-sensitive probes

• Structural approaches:

  • Cryo-EM structures in different conformational states

  • Hydrogen-deuterium exchange mass spectrometry to identify solvent-accessible regions

  • Molecular dynamics simulations to identify potential proton pathways

• Comparative analysis:

  • Structure-function comparison with well-characterized homologs from model organisms

  • Correlation between conserved residues and known proton pathways

  • Chimeric constructs combining domains from different species

These approaches would help determine whether nuoK forms part of the proton translocation pathway or plays a structural role in complex assembly.

How can thermostability of nuoK be assessed and compared to mesophilic homologs?

To assess and compare the thermostability of T. fusca nuoK with mesophilic homologs, researchers should employ:

• Thermal stability measurements:

  • Differential scanning calorimetry to determine melting temperatures (Tm)

  • Circular dichroism spectroscopy with temperature ramping

  • Thermal shift assays using environment-sensitive fluorescent dyes

  • Activity measurements at different temperatures

• Comparative sequence analysis:

  • Amino acid composition analysis (e.g., increased charged residues vs. decreased polar uncharged residues)

  • Identification of ion pairs, hydrogen bonds, and hydrophobic interactions

  • Analysis of proline residue distribution in loop regions

  • Comparison of aliphatic index and grand average of hydropathicity (GRAVY)

• Structural comparison:

  • Homology modeling based on solved structures

  • Molecular dynamics simulations at different temperatures

  • Analysis of structural rigidity and flexibility at different temperatures

• Protein engineering approaches:

  • Creation of chimeric proteins between thermophilic and mesophilic versions

  • Rational design of stabilizing mutations based on comparative analysis

  • Directed evolution for enhanced thermostability

T. fusca proteins generally exhibit adaptations typical of moderately thermophilic organisms, including increased charged residue content compared to mesophilic counterparts .

How can nuoK and other respiratory complex components from Thermobifida fusca contribute to bioenergy applications?

The respiratory complex components from T. fusca, including nuoK, offer several opportunities for bioenergy applications:

• Thermostable biofuel cells:

  • Development of enzymatic biofuel cells that operate at elevated temperatures

  • Increased reaction rates and reduced risk of microbial contamination

  • Greater stability in fluctuating environmental conditions

• Biomass conversion systems:

  • Integration with T. fusca's cellulolytic machinery for complete biomass processing systems

  • Coupling cellulose degradation to electricity generation

  • Development of consolidated bioprocessing approaches

• Electron transport engineering:

  • Creation of artificial electron transport chains with optimized efficiency

  • Designing electron transfer systems for biocatalytic processes

  • Development of hybrid systems combining components from different organisms

• Biosensors:

  • Creation of robust biosensors for environmental monitoring

  • Development of respiratory inhibitor detection systems

  • Design of thermostable electrochemical biosensors

The thermostability of T. fusca respiratory components offers advantages for applications requiring operation at elevated temperatures or in harsh environments.

What challenges exist in reconstituting functional membrane proteins like nuoK, and how can they be addressed?

Reconstitution of functional membrane proteins like nuoK presents several challenges:

• Expression challenges:

  • Low expression yields due to toxicity and limited membrane capacity

  • Protein misfolding and aggregation

  • Improper insertion into host membranes

  Solutions include:

  • Use of specialized expression strains (C41/C43)

  • Fusion partners that enhance membrane targeting

  • Controlled expression using tunable promoters

• Solubilization challenges:

  • Maintaining native structure during extraction from membranes

  • Selection of appropriate detergents

  • Protein destabilization during purification

  Solutions include:

  • Detergent screening to identify optimal solubilization conditions

  • Use of styrene-maleic acid copolymer (SMA) for native nanodiscs

  • Lipid-like peptide surfactants as alternatives to detergents

• Reconstitution challenges:

  • Achieving proper orientation in artificial membranes

  • Reconstituting multi-subunit complexes

  • Replicating native lipid environment

  Solutions include:

  • Nanodiscs with defined lipid composition

  • Cell-free expression with immediate incorporation into liposomes

  • Gradient-driven reconstitution for controlled orientation

• Functional assessment challenges:

  • Developing suitable activity assays for individual subunits

  • Distinguishing subunit-specific functions

  • Correlating structure with function

  Solutions include:

  • Development of chimeric proteins with reporter domains

  • Complementation assays in knockout systems

  • Advanced spectroscopic methods for localized measurements

How can comparative genomics provide insights into the evolution of respiratory complexes in Thermobifida fusca?

Comparative genomics approaches can reveal important insights about respiratory complex evolution in T. fusca:

• Phylogenetic analysis:

  • Construction of phylogenetic trees for respiratory complex subunits

  • Identification of evolutionary relationships across bacterial phyla

  • Analysis of co-evolution patterns between interacting subunits

• Genomic context analysis:

  • Examination of operon structures and gene clustering across related species

  • Identification of conserved gene arrangements versus lineage-specific rearrangements

  • Analysis of regulatory elements associated with respiratory genes

• Horizontal gene transfer assessment:

  • Detection of potential horizontal gene transfer events through GC content analysis

  • Codon usage pattern analysis across the genome

  • Identification of genomic islands containing respiratory components

• Adaptation signatures:

  • Identification of positive selection signatures in thermophilic lineages

  • Calculation of dN/dS ratios to detect sites under selective pressure

  • Correlation between genomic features and ecological niches

• Research approaches:

  • Whole genome comparison between T. fusca and related Actinobacteria

  • Analysis of the respiratory complex gene clusters across diverse bacterial phyla

  • Reconstruction of ancestral sequences to understand evolutionary trajectories

The T. fusca genome contains all typical respiratory complex components found in aerobic bacteria, organized in operons similar to those in other Actinobacteria, though with potential thermophilic adaptations .

What are the most promising research directions for understanding respiratory chain components in thermophilic bacteria?

Future research on respiratory chain components in thermophilic bacteria, including T. fusca nuoK, should focus on:

• Structural biology advancements:

  • High-resolution structures of complete respiratory complexes from thermophilic organisms

  • Time-resolved structural studies to capture intermediate states

  • Comparison of respiratory complex structures across temperature adaptations

• Systems biology approaches:

  • Metabolic flux analysis to understand respiratory chain function in cellular context

  • Integration of respiratory chain function with other cellular processes

  • Modeling of electron transport chain efficiency under different conditions

• Synthetic biology applications:

  • Design of minimal respiratory chains with optimized efficiency

  • Engineering of chimeric respiratory complexes with enhanced properties

  • Development of artificial electron transport systems for biotechnological applications

• Evolutionary biology questions:

  • Understanding the evolutionary trajectory of respiratory complexes

  • Elucidating the molecular basis of thermoadaptation in respiratory proteins

  • Investigating the co-evolution of respiratory complexes with cellular metabolism

• Biotechnological developments:

  • Harnessing thermostable respiratory components for bioenergy applications

  • Development of robust biosensors based on respiratory chain components

  • Engineering of electron transfer systems for sustainable chemistry applications

How might structural information about nuoK contribute to antimicrobial development?

Structural information about T. fusca nuoK could contribute to antimicrobial development through:

• Target identification:

  • Mapping unique structural features of bacterial respiratory complexes

  • Identification of essential residues for complex assembly and function

  • Determination of bacterial-specific features absent in mammalian counterparts

• Structure-based drug design:

  • Virtual screening against identified binding pockets

  • Fragment-based approaches to develop novel inhibitors

  • Design of peptidomimetics that disrupt complex assembly

• Resistance mechanisms:

  • Understanding structural basis of known resistance mechanisms

  • Identification of conserved regions less prone to resistance-conferring mutations

  • Design of multi-target inhibitors affecting multiple respiratory chain components

• Experimental approaches:

  • High-resolution structural determination of bacterial complex I

  • Structure-activity relationship studies with prototype inhibitors

  • Comparative structural analysis across diverse bacterial species

• Advantages of thermophilic models:

  • Enhanced structural stability facilitating crystallization

  • Higher resolution structural data for drug design

  • Thermostable proteins for binding assays and screening platforms

Respiratory chain complexes represent promising but underexploited targets for novel antimicrobial development, with structural information from model organisms like T. fusca potentially accelerating drug discovery efforts.