Recombinant Thermotoga petrophila ATP synthase subunit c (atpE)

What is Recombinant Thermotoga petrophila ATP synthase subunit c (atpE) and what are its basic properties?

Recombinant Thermotoga petrophila ATP synthase subunit c (atpE) is a membrane protein component of the F0 sector of ATP synthase, functioning in proton translocation across membranes during ATP synthesis. The full-length protein consists of 85 amino acids with the sequence MENLGDLAQGLALLGKYLGAGLCMGIGAIGPGIGEGNIGAHAMDAMARQPEMVGTITTRMLLADA VAETTGIYSLLIAFMILLVV . The protein is encoded by the atpE gene (UniProt ID: A5ILW7) and has several synonyms including ATP synthase F0 sector subunit c, F-type ATPase subunit c, and Lipid-binding protein . As a component from a hyperthermophilic organism, this protein exhibits remarkable thermostability, making it valuable for structural and functional studies of membrane proteins under extreme conditions. The commercially available recombinant form is typically produced with an N-terminal His-tag in E. coli expression systems .

How does Thermotoga petrophila differ from other Thermotoga species in terms of genomic and metabolic characteristics?

Thermotoga petrophila exhibits notable genomic and metabolic differences compared to other Thermotoga species like T. maritima, T. neapolitana, and Thermotoga sp. strain RQ2. While all four species share a core genome of approximately 1,470 ORFs with highly conserved central metabolism genes (including complete glycolytic, pentose phosphate, and Entner-Doudoroff pathways), T. petrophila shows significant distinctions in glucose utilization . Unlike other Thermotoga species, T. petrophila utilizes glucose to a lesser extent, which has been attributed to the absence of a specific glucose transporter (XylE2F2K2) that is present in other Thermotoga species . Instead, T. petrophila appears to acquire glucose through the XylE1F1K1 transporter, which primarily transports xylose in the other species . This metabolic divergence is reflected in gene expression patterns, where the XylR regulon is triggered by growth on glucose in T. petrophila . These differences support the designation of T. petrophila as a separate species within the Thermotogales and highlight the evolutionary adaptations in carbohydrate metabolism among hyperthermophilic bacteria.

What expression systems are recommended for producing functional Recombinant Thermotoga petrophila ATP synthase subunit c (atpE)?

For expressing Recombinant Thermotoga petrophila ATP synthase subunit c (atpE), E. coli expression systems represent the standard approach as demonstrated by commercial preparations . When designing expression systems, researchers should consider the hydrophobic nature of this membrane protein and its thermophilic origin. The recommended expression strategy involves using E. coli strains optimized for membrane protein expression, such as C41(DE3) or C43(DE3), combined with vectors containing N-terminal His-tags to facilitate purification . Expression should be conducted at lower temperatures (around 30°C) despite the thermophilic nature of the protein, as this reduces inclusion body formation and improves folding in the E. coli membrane. For purification, detergent-based extraction methods using mild detergents like n-dodecyl β-D-maltoside (DDM) help maintain protein functionality. The expression construct should include the complete 85-amino acid sequence to ensure proper folding and function . After purification, the protein can be reconstituted in lipid vesicles for functional studies, with 6% trehalose addition improving stability during storage .

How can researchers effectively analyze the thermostability mechanisms of Thermotoga petrophila ATP synthase subunit c compared to mesophilic homologs?

Analyzing thermostability mechanisms of Thermotoga petrophila ATP synthase subunit c requires a multi-faceted approach combining structural, biochemical, and computational methods. Researchers should first conduct comparative sequence analysis between T. petrophila atpE and mesophilic homologs to identify key amino acid differences, particularly focusing on hydrophobic core residues, ion pairs, and hydrogen bonding networks. Circular dichroism spectroscopy should be employed to measure protein unfolding at different temperatures (20-100°C), generating thermal denaturation curves for quantitative comparison. Differential scanning calorimetry provides complementary thermodynamic data on the protein's melting temperature and enthalpy changes during unfolding. For structural insights, X-ray crystallography or cryo-electron microscopy of the purified His-tagged protein can reveal atomic-level details of stabilizing interactions . Molecular dynamics simulations at elevated temperatures (80-100°C) can identify flexible regions and stabilizing interactions that persist under thermal stress. Hydrogen-deuterium exchange mass spectrometry provides experimental validation of computational predictions by measuring local conformational flexibility. For functional thermostability assessment, researchers should reconstitute the protein into liposomes and measure proton translocation activity at different temperatures, comparing activity retention between the thermophilic and mesophilic variants after heat treatment.

What are the critical considerations when designing mutagenesis studies to investigate structure-function relationships in Thermotoga petrophila ATP synthase subunit c?

When designing mutagenesis studies for Thermotoga petrophila ATP synthase subunit c, researchers must carefully consider several critical factors. First, amino acid selection should focus on conserved residues identified through multiple sequence alignment across species, particularly those in the transmembrane regions and the ion-binding site. The highly conserved nature of residues in positions involved in proton translocation makes them prime targets for alanine scanning mutagenesis . Second, researchers should consider the specific properties of T. petrophila's thermophilic environment when selecting mutations, particularly examining amino acids that differ from mesophilic homologs as these may contribute to thermostability. The glutamate residue essential for proton translocation should be investigated through conservative and non-conservative substitutions to dissect its precise role . When expressing mutant proteins, conditions should be optimized to ensure proper membrane insertion, potentially requiring adjustment of expression temperatures or inclusion of chaperones. Functional characterization should combine biochemical assays (ATP hydrolysis/synthesis) with biophysical methods (thermostability measurements, structural analyses). For comprehensive analysis, researchers should create a library of mutations spanning the entire 85-amino acid sequence while prioritizing regions with predicted functional importance . Coupling mutations with crosslinking studies can provide insights into subunit c interactions within the c-ring oligomer of the ATP synthase complex.

How can researchers effectively analyze the oligomeric structure of the c-ring from Thermotoga petrophila ATP synthase and its impact on function?

Analyzing the oligomeric structure of the c-ring from Thermotoga petrophila ATP synthase requires an integrated structural biology approach. Researchers should begin with expression and purification of the His-tagged recombinant atpE protein using conditions that maintain the native oligomeric state, potentially including mild detergents like DDM or digitonin during extraction from E. coli membranes . For initial structural characterization, blue native polyacrylamide gel electrophoresis (BN-PAGE) can determine the approximate molecular weight of the c-ring complex. To determine the precise number of c-subunits in the ring, researchers should employ high-resolution structural techniques including cryo-electron microscopy, which has advantages for membrane protein complexes, or X-ray crystallography following successful crystallization trials. Cross-linking experiments using bi-functional reagents followed by mass spectrometry can provide complementary data on subunit-subunit interactions within the ring. Atomic force microscopy of reconstituted c-rings in lipid bilayers can yield information about topography and mechanical properties. For functional assessment, the purified c-rings should be reconstituted with other ATP synthase components to measure proton translocation efficiency and ATP synthesis rates. The c-ring stoichiometry should be correlated with the physiological growth temperature of T. petrophila to understand thermal adaptation mechanisms. Researchers should also compare the findings with c-rings from other Thermotoga species to identify species-specific adaptations in this hyperthermophilic genus .

What are the optimal conditions for reconstituting Thermotoga petrophila ATP synthase subunit c into liposomes for functional studies?

Reconstituting Thermotoga petrophila ATP synthase subunit c into liposomes requires careful optimization of multiple parameters to ensure functional membrane insertion. Researchers should begin with purified His-tagged atpE protein (>90% purity as confirmed by SDS-PAGE) in a Tris/PBS-based buffer containing 6% trehalose . The optimal lipid composition should include a mixture of E. coli polar lipids and phosphatidylcholine (7:3 ratio) to mimic bacterial membrane characteristics while providing stability. For thermophilic proteins like T. petrophila atpE, incorporating archaeal lipids such as diphytanyl phospholipids can enhance functional stability at elevated temperatures (70-80°C). The protein-to-lipid ratio should be optimized, typically starting with 1:100 (w/w) and testing ratios between 1:50 and 1:200. The reconstitution protocol should employ a detergent removal method using Bio-Beads SM-2 or controlled dialysis, with the process conducted at 30°C to facilitate proper protein integration while preventing aggregation. Buffer conditions during reconstitution should maintain pH 7.5-8.0 (using Tris or HEPES buffer) with 100-150 mM KCl to establish ion gradients for functional assays . Prior to functional testing, researchers should verify successful reconstitution through freeze-fracture electron microscopy or fluorescence recovery after photobleaching (FRAP) to confirm protein integration and lateral mobility. Functional assessment should include proton translocation assays using pH-sensitive fluorescent dyes (ACMA or pyranine) and ATP synthesis measurements when co-reconstituted with other ATP synthase components.

What approaches should be used to study the interaction between Thermotoga petrophila ATP synthase subunit c and other components of the ATP synthase complex?

Studying interactions between Thermotoga petrophila ATP synthase subunit c and other complex components requires multiple complementary approaches. Researchers should begin with co-immunoprecipitation experiments using antibodies against the His-tag of recombinant atpE to pull down interacting partners from T. petrophila lysates, followed by mass spectrometry identification . For reconstitution studies, researchers should co-express atpE with other ATP synthase components in E. coli, focusing particularly on interactions with a-subunit and peripheral stalk components. Surface plasmon resonance (SPR) or bio-layer interferometry can quantify binding kinetics between purified atpE and other subunits, with the His-tagged protein immobilized on sensor chips. Chemical cross-linking coupled with mass spectrometry should be employed to map interaction interfaces, using membrane-permeable cross-linkers with varying spacer lengths to identify proximity relationships. Förster resonance energy transfer (FRET) experiments using fluorescently labeled subunits can detect interactions in reconstituted systems under near-native conditions. For structural studies, cryo-electron microscopy of the assembled complex provides visualization of subunit arrangements, while hydrogen-deuterium exchange mass spectrometry identifies regions of atpE that show altered solvent accessibility upon complex formation. Computational approaches including molecular docking and molecular dynamics simulations can predict interaction interfaces and binding energetics. Functional validation through site-directed mutagenesis of predicted interface residues should be performed to confirm their importance in complex assembly and activity. Comparative analysis with subunit interactions in other Thermotoga species can highlight conserved and species-specific interaction networks .

What techniques should be employed to measure the proton translocation activity of reconstituted Thermotoga petrophila ATP synthase subunit c?

Measuring proton translocation activity of reconstituted Thermotoga petrophila ATP synthase subunit c requires specialized techniques that monitor ion movement across membranes. The primary approach involves reconstituting the purified His-tagged atpE protein into liposomes containing pH-sensitive fluorescent dyes . Researchers should prepare proteoliposomes using the optimized reconstitution protocol, incorporating either ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine as internal pH indicators. For ACMA-based assays, researchers should establish a pH gradient across the membrane using buffers of different pH or by activating ATP synthase, then monitor fluorescence quenching as protons are transported. Real-time measurements should be conducted at temperatures ranging from 37°C to 80°C to assess thermostability of function, with particular attention to activity at T. petrophila's optimal growth temperature. Solid-supported membrane (SSM) electrophysiology represents an alternative approach, allowing direct electrical measurement of charge translocation through the immobilized proteoliposomes. For comprehensive analysis, researchers should combine these methods with ATP synthesis/hydrolysis assays to correlate proton translocation with enzymatic activity. The effect of ionophores (like FCCP) and specific inhibitors (like DCCD) should be tested to verify the specificity of measured activities. Comparison with reconstituted c-subunits from mesophilic organisms under identical conditions provides valuable insights into thermoadaptation of function. Data analysis should include calculation of proton translocation rates, determination of activation energies from Arrhenius plots, and assessment of pH dependence to characterize the functional properties of T. petrophila atpE.

How should researchers analyze transcriptomic data to understand the regulation of atpE expression in Thermotoga petrophila compared to other Thermotoga species?

Analyzing transcriptomic data for atpE regulation in Thermotoga petrophila requires a systematic approach to identify expression patterns and regulatory mechanisms. Researchers should first normalize raw transcriptomic data (RNA-Seq or microarray) using appropriate statistical methods to account for technical and biological variation across datasets. Differential expression analysis should compare atpE expression in T. petrophila versus other Thermotoga species (T. maritima, T. neapolitana, and Thermotoga sp. strain RQ2) under identical growth conditions, using fold-change thresholds and adjusted p-values to identify significant differences . Co-expression analysis should identify genes that show correlated expression patterns with atpE, potentially revealing functional relationships or shared regulatory mechanisms. For regulatory element identification, researchers should perform promoter analysis of the atpE gene across species, scanning upstream regions for conserved binding motifs that might interact with transcription factors. Gene set enrichment analysis can identify biological pathways and functions associated with differentially expressed genes, placing atpE regulation in broader metabolic context. Researchers should particularly investigate correlations between atpE expression and genes involved in energy metabolism or stress response. The comparison with expression data from the table in search result provides valuable reference points for expression changes under different conditions. Time-course experiments analyzing expression during growth phases or in response to environmental stressors (temperature shifts, nutrient limitation) can reveal dynamic regulation patterns. Integration with proteomic data adds another dimension, allowing correlation between transcript levels and protein abundance to identify post-transcriptional regulation.

How can researchers effectively interpret structural data of Thermotoga petrophila ATP synthase subunit c to explain its thermostability and functional properties?

Interpreting structural data of Thermotoga petrophila ATP synthase subunit c requires careful analysis of multiple structural features that contribute to thermostability and function. Researchers should begin by examining the amino acid composition of the 85-residue protein, quantifying the prevalence of thermostabilizing features such as increased hydrophobic core packing, additional salt bridges, and reduced conformational flexibility . Secondary structure analysis should focus on the stability of alpha-helical transmembrane regions, comparing helix propensities and length with mesophilic homologs. Researchers must analyze the ion-binding site microenvironment, particularly examining the coordination geometry and electrostatic environment around the conserved proton-binding glutamate residue essential for function. Oligomeric interfaces between subunit c molecules within the c-ring merit special attention, as inter-subunit interactions significantly contribute to thermostability. When examining structural data, researchers should calculate the accessible surface area and buried hydrophobic surface to quantify packing efficiency. Molecular dynamics simulations at elevated temperatures (80-100°C) can reveal dynamic properties not evident in static structures, identifying regions with thermal motion and stabilizing interactions that persist at high temperatures. For comprehensive interpretation, researchers should correlate structural features with experimental thermostability data (melting temperatures, activity retention after heat treatment) and examine how specific amino acid differences from the sequence MENLGDLAQGLALLGKYLGAGLCMGIGAIGPGIGEGNIGAHAMDAMARQPEMVGTITTRMLLADA VAETTGIYSLLIAFMILLVV contribute to observed properties . Comparative analysis with structures from mesophilic organisms can highlight adaptations specific to the thermophilic lifestyle.