Recombinant Thermus thermophilus NADH-quinone oxidoreductase subunit K (nuoK)

What is the genomic context of nuoK in Thermus thermophilus?

The genome of T. thermophilus HB27 consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27, with 2,218 identified putative genes . While the search results don't specifically detail the nuoK genomic location, it would be part of the nuo operon encoding the 14 subunits of bacterial NADH-quinone oxidoreductase (NDH-1). To determine the precise location, researchers should perform whole genome analysis using next-generation sequencing, followed by annotation using homology-based approaches comparing with the well-characterized NDH-1 complex from other bacteria.

How does the structure of T. thermophilus NADH-quinone oxidoreductase compare to other bacterial systems?

The bacterial NADH-quinone oxidoreductase in T. thermophilus HB-8 consists of 14 subunits, which is structurally simpler than the mammalian mitochondrial enzyme (complex I) that contains more than 40 subunits . The nuoK subunit is part of the membrane domain and likely contributes to the proton translocation pathway. Comparative structural analysis using cryo-EM or X-ray crystallography would reveal thermostability-related structural adaptations in T. thermophilus nuoK compared to mesophilic homologs.

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

While T. thermophilus is naturally competent for transformation with either chromosomal or plasmid DNA , expression of its membrane proteins like nuoK presents challenges. E. coli-based expression systems have been successfully used for other T. thermophilus proteins, as demonstrated with NusG . For nuoK expression, a methodological approach would include:

• Codon optimization for the host system

• Use of strong inducible promoters (T7 or tac)

• Addition of fusion tags (His6, MBP) to facilitate purification

• Growth at lower temperatures (18-25°C) to improve membrane protein folding

• Supplementation with specific lipids that might be required for proper folding

For particularly challenging membrane proteins, cell-free expression systems or specialized membrane protein expression hosts like C41/C43(DE3) E. coli strains should be considered.

What purification strategies yield the highest activity for recombinant nuoK?

Purification of membrane proteins like nuoK requires careful detergent selection. A methodological workflow would include:

• Initial membrane isolation by ultracentrifugation

• Solubilization screening with different detergents (DDM, LMNG, digitonin)

• Heat treatment at 65°C to exploit thermostability and remove contaminants

• Affinity chromatography using engineered tags

• Size exclusion chromatography to ensure homogeneity

• Activity assessment in proteoliposomes or nanodiscs

For thermostable proteins from T. thermophilus, heat treatment becomes a powerful purification step that takes advantage of their exceptional stability while eliminating most E. coli proteins .

How can photoaffinity labeling be adapted to study nuoK interactions within the NADH-quinone oxidoreductase complex?

Drawing from approaches used with the PSST subunit of complex I, photoaffinity labeling can be a powerful technique for studying nuoK interactions. The PSST subunit was successfully labeled using (trifluoromethyl)diazirinyl[3H]pyridaben as a photoaffinity ligand, which combined high inhibitor potency, a suitable photoreactive group, and tritium labeling . For nuoK studies, a methodological workflow would include:

• Design of photoaffinity probes targeting regions where nuoK interfaces with other subunits

• Validation of probe specificity through competitive binding assays

• Photolabeling of intact complex under varying conditions

• Subunit isolation and mass spectrometry analysis

• Cross-validation using site-directed mutagenesis of identified interaction sites

This approach would reveal the spatial arrangement of nuoK relative to other subunits and identify residues critical for subunit interactions.

What methodological approaches can resolve conflicting data about nuoK function across different experimental conditions?

Similar to observations with the NusG transcription factor, which shows species-specific functional differences (Tth NusG slows down transcript elongation while E. coli NusG increases elongation rate) , nuoK might exhibit context-dependent functions. To resolve such conflicts, researchers should:

• Establish standardized assay conditions that account for temperature, pH, and ionic strength variations

• Perform chimeric protein studies swapping domains between nuoK from different species

• Conduct in vivo complementation assays to determine functional equivalence

• Use reconstituted systems of increasing complexity to identify context-dependent effects

• Apply single-molecule techniques to detect heterogeneity in function that might be masked in bulk assays

This systematic approach would help identify whether functional differences are intrinsic to the protein or result from experimental conditions.

How does the thermostability of nuoK affect its structural dynamics during the catalytic cycle?

T. thermophilus proteins exhibit exceptional thermostability, necessary for function at the organism's optimal growth temperature (65-70°C) . For nuoK, this thermostability likely impacts its conformational dynamics. A methodological investigation would include:

• Hydrogen-deuterium exchange mass spectrometry at different temperatures

• Molecular dynamics simulations comparing behavior at mesophilic versus thermophilic temperatures

• Temperature-dependent EPR spectroscopy to monitor specific labeled residues

• Time-resolved structural studies using temperature-jump techniques

• Comparative analysis with mesophilic homologs to identify thermostability-conferring features

These approaches would reveal how nuoK maintains functional flexibility while retaining structural integrity at high temperatures.

What genetic tools can be employed to study nuoK function in vivo in T. thermophilus?

T. thermophilus is amenable to genetic manipulation due to its natural competence for transformation . A counterselectable marker system using pheS has been developed that allows introduction of unmarked deletions and point mutations . For nuoK studies, researchers could:

• Generate conditional knockouts using the p-Cl-Phe counterselection system

• Introduce site-specific mutations to test structure-function hypotheses

• Create reporter gene fusions to monitor expression and localization

• Develop CRISPR-Cas9 systems adapted for high-temperature function

• Perform complementation studies with heterologous nuoK variants

The transformation efficiency of T. thermophilus facilitates these genetic manipulations, allowing for rapid strain construction and phenotypic analysis.

The NADH-quinone oxidoreductase couples electron transfer from NADH to quinone with proton translocation across the membrane . While the search results don't specifically detail nuoK's role, its position in the membrane domain suggests involvement in proton translocation. To investigate this coupling mechanism:

• Introduce mutations in conserved charged residues potentially forming proton transfer pathways

• Measure electron transfer rates (NADH oxidation) and proton pumping efficiency simultaneously

• Perform molecular dynamics simulations focusing on water dynamics within potential proton channels

• Use pH-sensitive fluorescent probes to track proton movement in reconstituted systems

• Apply electrophysiological methods to measure proton translocation in single-complex studies

This would establish whether nuoK forms part of the proton translocation pathway or plays a structural role supporting the coupling mechanism.

How does the inhibitor sensitivity profile of T. thermophilus NADH-quinone oxidoreductase compare to other bacterial and mitochondrial systems?

Complex I and bacterial NDH-1 are sensitive to diverse inhibitors including rotenone, piericidin A, bullatacin, and pyridaben . To characterize the inhibitor sensitivity profile with respect to nuoK:

• Perform comparative inhibition studies with a panel of known inhibitors

• Use photoaffinity labeling with inhibitor analogs to identify binding sites within or near nuoK

• Generate resistant mutants and map mutations to specific subunits

• Conduct molecular docking and dynamics simulations to model inhibitor binding

• Perform structure-activity relationship studies with modified inhibitors

This approach would reveal whether nuoK contributes to inhibitor binding sites and how thermophilic adaptations might affect inhibitor interactions.

What strategies can overcome the challenges of working with thermostable membrane proteins like nuoK?

Working with thermostable membrane proteins presents unique challenges that require specialized approaches:

• Buffer optimization to maintain stability at lower temperatures during purification

• Detergent screening considering both extraction efficiency and protein stability

• Specialized thermostable chromatography resins and equipment

• Activity assays adapted to function at elevated temperatures

• Modified reconstitution protocols using thermostable lipids

The exceptional stability of T. thermophilus proteins can be leveraged for more rigorous purification conditions, potentially yielding higher purity preparations .

How can collaborative approaches enhance research on nuoK and the NADH-quinone oxidoreductase complex?

Research on complex systems like NADH-quinone oxidoreductase benefits from multidisciplinary collaboration:

• Structural biologists providing high-resolution structures

• Biochemists characterizing enzymatic activities

• Molecular biologists developing genetic tools

• Biophysicists applying spectroscopic and single-molecule techniques

• Computational scientists performing simulations and systems modeling

The combination of these approaches provides complementary insights that no single technique could achieve independently, facilitating a comprehensive understanding of nuoK function within the larger complex.