Recombinant Thermus thermophilus NADH-quinone oxidoreductase subunit C (nuoC)

What is the functional role of NuoC in the NADH-quinone oxidoreductase complex?

NuoC (Nqo5 in T. thermophilus) functions as a structural component within the peripheral arm of the NADH-quinone oxidoreductase complex (NDH-1). Unlike most subunits in the peripheral arm, NuoC does not directly bind any cofactors, yet it plays a crucial role in maintaining the structural integrity of the complex . The subunit forms part of the electron transfer pathway from NADH to quinone, although it does not directly participate in electron transfer. Its primary function appears to be maintaining proper spatial arrangement of other subunits through specific protein-protein interactions, particularly via conserved acidic residues that form ionic bonds with adjacent subunits . Experimental evidence shows that mutations in key residues of NuoC result in compromised complex assembly and diminished enzymatic activity, highlighting its essential structural role in the functional architecture of NDH-1 .

How does the structure of NuoC in T. thermophilus differ from its homologs in other organisms?

In T. thermophilus, NuoC exists as a distinct subunit called Nqo5, whereas in organisms like Escherichia coli and Bacteroides fragilis, NuoC and NuoD are fused into a single polypeptide (NuoCD) . The NuoC subunit shares homology with the N-terminal domain of HycE and its paralogue HyfG in E. coli . Structural studies of T. thermophilus complex I reveal that NuoC (Nqo5) forms a compact structure with several conserved α-helices, including a critical third α-helix containing highly conserved acidic residues (Glu-117/Glu-140 in E. coli and Asp-120/Asp-143 in E. coli) . These residues form important ionic interactions with residues in adjacent subunits. The thermostable nature of T. thermophilus proteins enables structural stability at temperatures up to 65°C, making T. thermophilus NuoC particularly valuable for structural studies and in vitro protein synthesis experiments .

What expression systems are most effective for producing recombinant T. thermophilus NuoC?

For the expression of recombinant T. thermophilus NuoC, E. coli-based expression systems have proven effective despite the phylogenetic distance between these organisms . The optimal methodology involves:

• Gene optimization: Codon optimization for E. coli expression

• Vector selection: pET-series vectors with T7 promoter systems

• Host strain selection: BL21(DE3) or Rosetta strains for enhanced expression of rare codons

• Growth conditions: Initial growth at 37°C followed by induction at lower temperatures (18-25°C)

• Induction parameters: 0.1-0.5 mM IPTG for 4-16 hours

The thermostable nature of T. thermophilus proteins allows for heat treatment purification steps (60-65°C for 10-15 minutes) to remove most E. coli proteins while retaining NuoC activity . This property significantly simplifies purification protocols and yields highly pure protein preparations. When expressing the entire complex, co-expression strategies using polycistronic constructs or multiple compatible plasmids have shown success in reconstituting functional protein assemblies .

How do the conserved carboxyl residues in NuoC contribute to NDH-1 assembly and function?

Three highly conserved carboxyl residues in the NuoC segment—Glu-138, Glu-140, and Asp-143 in E. coli (corresponding to residues in T. thermophilus Nqo5)—play pivotal roles in NDH-1 structure and function . Site-directed mutagenesis studies revealed that mutations of these residues severely impair both enzyme assembly and activity:

Structural analysis revealed that Glu-140 (Glu-117 in T. thermophilus) forms critical ionic interactions with Arg-600 (Arg-409 in T. thermophilus) of the NuoD segment at a distance of approximately 2.8Å . Additionally, this residue interacts with Arg-560 (Lys-369 in T. thermophilus) at a distance of 3.1-3.3Å . These interactions appear to stabilize the interface between NuoC and NuoD segments, ensuring proper assembly of the peripheral arm. Disruption of these ionic bonds through mutation results in destabilization of the entire complex, highlighting their essential structural role beyond simple electrostatic interactions .

What methodological approaches can resolve contradictory data regarding NuoC function in different temperature environments?

Research has revealed an apparent contradiction: while T. thermophilus grows optimally at elevated temperatures, reconstituted translation systems containing NuoC and other components remain functional at temperatures as low as 37°C, well below the organism's minimal growth temperature . To resolve such contradictory findings, the following methodological approaches are recommended:

• Comparative activity assays across temperature ranges: Measure enzymatic activities (dNADH-K₃Fe(CN)₆ reductase, dNADH-DB reductase, and dNADH oxidase) at temperatures ranging from 30-70°C using the following protocol:

  • Prepare membrane samples (80 μg protein/ml) in appropriate buffer (10 mM potassium phosphate, pH 7.0, 1 mM EDTA, 10 mM KCN)

  • Add substrate and monitor activity spectrophotometrically (420 nm for K₃Fe(CN)₆, 340 nm for dNADH)

• Structural stability assessment: Use differential scanning calorimetry (DSC) and circular dichroism (CD) to monitor thermal unfolding transitions at different temperatures.

• Functional reconstitution experiments: Perform in vitro reconstitution of NDH-1 complexes at different temperatures using purified components to identify temperature-dependent assembly factors .

• Polyamine dependence analysis: Investigate the role of polyamines (particularly tetraamine) in stabilizing functional complexes across temperature ranges, as they have been shown to be required for translation at both high and low temperatures .

The apparent contradiction might be explained by different requirements for in vivo growth versus in vitro protein function, or by the presence of additional cellular factors that influence temperature dependence in the living organism but are absent in reconstituted systems .

How can structural data from T. thermophilus NuoC inform mutagenesis studies to explore energy coupling mechanisms?

The available structural data for T. thermophilus Nqo5 (NuoC) provides an excellent foundation for rational mutagenesis approaches to explore energy coupling mechanisms in NDH-1. Effective research strategies include:

• Structure-guided mutagenesis targeting conserved residues: Based on the crystal structure of T. thermophilus NDH-1 (PDB codes: 3FUG, 3IAS), target the following:

  • Conserved acidic residues in the third α-helix (particularly Glu-117/Glu-140 and Asp-120/Asp-143)

  • Residues at interfaces with other subunits, especially those forming salt bridges

• Chimeric protein construction: Create chimeric proteins by swapping domains between NuoC and its paralogue HycE N-terminal domain to investigate evolutionary relationships and functional convergence .

• Proton pumping assays: Assess the impact of mutations on proton translocation using:

  • Membrane potential measurements with oxonol VI (monitoring absorbance changes at 630-603 nm)

  • Proton pump activity assessment via ACMA fluorescence quenching

• Combining structural and functional approaches:

  • Map mutations onto the 3D structure to visualize spatial relationships

  • Correlate structural changes with alterations in energy coupling efficiency

  • Use molecular dynamics simulations to predict effects of mutations on structural dynamics

This integrated approach can help establish structure-function relationships and potentially identify residues involved in long-range conformational changes necessary for energy coupling between electron transport and proton translocation .

What are the optimal conditions for soluble expression of recombinant T. thermophilus NuoC?

Achieving high yields of soluble recombinant T. thermophilus NuoC requires careful optimization of expression conditions. The following protocol has been found effective through systematic testing:

• Construct design:

  • Include an N-terminal His₆-tag for purification

  • Consider fusion partners (SUMO or MBP) to enhance solubility if initial expression yields are low

  • Ensure the coding sequence is codon-optimized for the expression host

• Expression conditions:

  • Host: E. coli BL21(DE3) or Rosetta(DE3) for rare codon expression

  • Media: LB or rich media (such as Terrific Broth) with appropriate antibiotics

  • Growth temperature: 37°C until OD₆₀₀ reaches 0.6-0.8

  • Induction: 0.2-0.5 mM IPTG

  • Post-induction growth: 18°C for 16-20 hours to maximize soluble protein yield

• Cell lysis and initial purification:

  • Resuspend cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

  • Lyse cells by sonication or high-pressure homogenization

  • Heat treatment (60°C for 15 minutes) to exploit the thermostability of T. thermophilus proteins and remove host contaminants

  • Clarify by centrifugation (20,000 × g, 30 minutes)

This approach typically yields 20-30 mg of soluble NuoC per liter of culture with >90% purity after heat treatment and initial Ni-NTA chromatography .

How can researchers effectively characterize the functional integrity of purified recombinant NuoC?

To ensure that purified recombinant NuoC maintains its structural and functional integrity, multiple complementary characterization techniques should be employed:

• Biochemical characterization:

  • Size exclusion chromatography to assess oligomeric state

  • Thermal stability analysis using differential scanning fluorimetry (DSF)

  • Limited proteolysis to evaluate proper folding and stability

• Functional reconstitution:

  • Assembly with other purified subunits to form functional subcomplexes or the entire NDH-1 complex

  • Assessment of reconstitution efficiency through Blue Native PAGE

  • Measurement of NADH:ubiquinone oxidoreductase activity in the reconstituted complex

• Structural validation:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure elements

  • Comparison with reference spectra from native protein

  • FTIR analysis for additional structural information

• Interaction studies:

  • Isothermal titration calorimetry (ITC) or microscale thermophoresis (MST) to measure binding to known interaction partners

  • Pull-down assays to confirm interactions with adjacent subunits in the complex

Proper functional characterization should include reconstitution into proteoliposomes to measure membrane potential generation and proton pumping activity, which are key indicators of NuoC's correct integration into a functional complex .

What are the unresolved questions about the evolutionary relationship between NuoC and HycE?

The evolutionary relationship between NuoC and the N-terminal domain of HycE presents several unresolved questions that merit further investigation:

• Structural homology vs. functional divergence: Despite structural similarities, NuoC and the N-terminal domain of HycE participate in distinct molecular processes. Research should address how similar protein scaffolds evolved to support different functions in respiratory and hydrogenase complexes .

• Fusion events in protein evolution: In E. coli, NuoC and NuoD are fused, whereas they exist as separate proteins in many other organisms. Similarly, the N-terminal domain of HycE is fused to the catalytic domain. Research questions include:

  • What evolutionary pressures drove these fusion events?

  • How do these fusion events affect assembly pathways and complex stability?

  • Can the functional significance of these fusions be demonstrated experimentally?

• Methodological approaches to address these questions:

  • Phylogenetic analysis across diverse bacterial and archaeal lineages

  • Construction and functional testing of split and fused protein variants

  • Ancestral sequence reconstruction to test hypotheses about evolutionary trajectories

Recent experiments involving domain splitting of HycE and NuoCD provide preliminary insights, suggesting that the domain fusions may have evolved to enhance assembly efficiency and complex stability rather than for direct catalytic functions .

How can advanced spectroscopic techniques enhance our understanding of NuoC's role in complex I assembly?

Advanced spectroscopic techniques offer powerful tools for investigating NuoC's role in complex I assembly at unprecedented resolution:

• Single-molecule FRET (smFRET) can track conformational changes during assembly:

  • Label purified NuoC and potential interaction partners with appropriate FRET pairs

  • Monitor dynamic assembly processes in real-time

  • Identify intermediate assembly states that may be missed in bulk experiments

• Cryo-electron microscopy (Cryo-EM) combined with cross-linking mass spectrometry:

  • Visualize assembly intermediates at near-atomic resolution

  • Identify structural changes during complex formation

  • Map the positions of key residues, particularly the conserved acidic residues in helix 3

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Map regions of NuoC that undergo structural changes upon interaction with other subunits

  • Identify protected regions that form stable interfaces

  • Correlate with mutagenesis data to validate functional importance of specific regions

• Time-resolved NMR spectroscopy:

  • Monitor structural changes in real-time during assembly

  • Identify transient interactions that may guide the assembly process

  • Map the dynamics of specific residues during complex formation

Implementation of these techniques requires careful experimental design, including:

• Expression of isotopically labeled proteins for NMR studies

• Site-specific labeling for FRET experiments

• Sample preparation optimization for Cryo-EM

• Development of reconstitution systems that allow time-resolved monitoring of assembly

This integrated spectroscopic approach promises to reveal the dynamic process of complex I assembly and the specific role of NuoC in this process.