Recombinant Bifidobacterium longum subsp. infantis ATP synthase subunit c (atpE)

What is the structural composition of B. longum subsp. infantis ATP synthase subunit c (atpE)?

The ATP synthase subunit c (atpE) from Bifidobacterium longum subsp. infantis is a small membrane protein consisting of 76 amino acids with the following sequence: MDIITLAEVAGNLSVIGYGIGTLGPGIGLGILFGKAMESTARQPEMSGKIQTIMFIGLALVEVLALIGFVAALIIR . This protein belongs to the F-type ATPase family and functions as part of the F₀ membrane domain of ATP synthase. In bacterial ATP synthases, the subunit c forms an oligomeric ring structure that is essential for proton translocation and the energy conversion process. The protein contains hydrophobic regions that facilitate its integration into the membrane, where it participates in the rotary mechanism of ATP synthesis.

How does recombinant atpE differ from native atpE in terms of functionality?

Recombinant atpE proteins are engineered versions expressed in heterologous systems such as E. coli, yeast, baculovirus, or mammalian cells . While they maintain the primary sequence of the native protein, several factors can affect their functionality:

• Expression system influences: Different expression systems (E. coli, yeast, etc.) may introduce subtle variations in post-translational modifications that can affect protein folding and activity.

• Tag additions: Recombinant versions often contain tags (such as His-tag) that facilitate purification but may influence protein behavior. For example, N-terminal His-tagged atpE proteins may exhibit altered membrane insertion or oligomerization properties .

• Self-assembly capabilities: Research has shown that recombinant ATP synthase subunit c can self-assemble into annular structures even in the absence of other subunits, suggesting that the ring-forming ability is encoded in its primary structure . This property is maintained in the recombinant form.

When using recombinant atpE in research, it's essential to validate that the protein retains its native oligomerization properties and ability to participate in proton translocation.

What are the recommended storage and handling conditions for recombinant atpE?

For optimal stability and activity of recombinant Bifidobacterium longum subsp. infantis ATP synthase subunit c (atpE), follow these evidence-based handling recommendations:

Storage conditions:

• Store lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, aliquot the protein to avoid repeated freeze-thaw cycles

• For short-term use, store working aliquots at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• The default recommended final concentration of glycerol is 50%

Critical considerations:

• Repeated freezing and thawing significantly reduces protein activity and should be avoided

• The protein is typically provided in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0, which helps maintain stability during storage

What are the optimal expression systems for producing functional recombinant atpE?

The choice of expression system for recombinant Bifidobacterium longum subsp. infantis ATP synthase subunit c (atpE) depends on your specific research requirements. Based on available data, several expression systems have been successfully used:

For functional studies of atpE, E. coli expression systems have proven effective and offer the advantage of in vivo biotinylation when the protein is co-expressed with BirA ligase . The E. coli system is particularly suitable for structural studies and has successfully produced recombinant ATP synthase subunit c that demonstrates self-assembly into oligomeric rings, similar to the native protein .

When expressing atpE, consider the following methodological aspects:

• Use low-to-moderate induction conditions to prevent inclusion body formation

• Include membrane-mimetic environments during purification

• Select appropriate detergents that maintain the protein's native oligomeric state

How can researchers verify the correct oligomerization of recombinant atpE?

Verifying the correct oligomerization of recombinant ATP synthase subunit c (atpE) is critical for functional studies. Research has shown that subunit c can self-assemble into annular structures even in the absence of other ATP synthase components . Here are methodological approaches to verify proper oligomerization:

Analytical techniques:

• Blue Native PAGE (BN-PAGE):

  • Separates native protein complexes based on size

  • Preserves quaternary structures during electrophoresis

  • Can reveal the presence of c-rings of appropriate molecular weight

• Size Exclusion Chromatography (SEC):

  • Provides information about the hydrodynamic radius of the complex

  • Can distinguish between monomeric and oligomeric states

  • Should show a peak corresponding to the expected molecular weight of the c-ring (approximately 10-11 subunits for bacterial ATP synthases)

• Transmission Electron Microscopy (TEM):

  • Direct visualization of ring structures

  • Negative staining can enhance contrast of membrane protein complexes

  • Can confirm annular architecture with dimensions consistent with c-rings

• Atomic Force Microscopy (AFM):

  • Provides topographical information of the protein complex

  • Can be performed in near-native conditions

  • Allows measurement of ring dimensions and subunit arrangement

• Cross-linking coupled with mass spectrometry:

  • Chemical cross-linking can capture subunit-subunit interactions

  • Mass spectrometry identifies cross-linked peptides

  • Provides evidence of spatial proximity consistent with c-ring formation

When analyzing atpE oligomerization, keep in mind that the c-ring typically contains 10-11 subunits in E. coli and may vary in other species . The ability to form these rings is determined by the primary structure of the protein, highlighting the importance of maintaining the native sequence in recombinant versions.

What are the best methods for studying proton translocation function of atpE in vitro?

Studying the proton translocation function of recombinant ATP synthase subunit c (atpE) requires reconstitution of the protein into membrane-mimetic systems. Here are methodological approaches for investigating this critical function:

Liposome reconstitution approaches:

• Preparation of proteoliposomes:

  • Purified recombinant atpE should be reconstituted into liposomes composed of bacterial phospholipids

  • Maintain a lipid-to-protein ratio that allows formation of functional c-rings

  • Use detergent removal methods (dialysis, Bio-Beads, or gel filtration) to incorporate protein into liposomes

• pH-sensitive fluorescent probes:

  • Incorporate pH-sensitive fluorescent dyes (ACMA, pyranine) into proteoliposomes

  • Monitor fluorescence changes upon establishment of a proton gradient

  • Quantify proton translocation rates under various conditions

• Patch-clamp electrophysiology:

  • For direct measurement of proton currents through reconstituted atpE channels

  • Provides detailed kinetic information about proton translocation

  • Can be combined with site-directed mutagenesis to identify key residues

Important experimental controls:

• Include ionophores (e.g., CCCP) to collapse proton gradients as negative controls

• Compare wild-type atpE with known non-functional mutants

• Verify that proton translocation is coupled to ATP synthesis/hydrolysis in complete systems

When studying the specific contribution of B. longum subsp. infantis atpE to proton translocation, researchers should consider the metabolic context of this organism, which produces acetate and can engage in cross-feeding interactions with other gut bacteria like Eubacterium rectale . These ecological relationships may have influenced the evolutionary adaptations of the ATP synthase complex in B. longum.

How does atpE contribute to metabolic cross-feeding between B. longum and other gut microbiota?

ATP synthase subunit c (atpE) plays a crucial role in energy metabolism of Bifidobacterium longum, which influences its interactions with other gut microbiota members. Research indicates that B. longum subsp. longum NCC2705, closely related to B. longum subsp. infantis, engages in mutual cross-feeding interactions with butyrate-producing bacteria such as Eubacterium rectale .

Metabolic relationship framework:

• Energy harvesting mechanisms:

  • The ATP synthase complex, of which atpE is an essential component, generates ATP through oxidative phosphorylation

  • This energy production supports B. longum's role as an acetate producer and arabinose substituent degrader of AXOS (arabinoxylan oligosaccharides)

  • The acetate produced by B. longum serves as a substrate for butyrate production by E. rectale

• Experimental evidence of cross-feeding:

  • Studies have monitored gene expression changes in both organisms during co-culture

  • Reverse transcription-quantitative PCR (RT-qPCR) has been used to assess differential expression of genes related to AXOS consumption and metabolite production

  • Growth conditions for studying these interactions typically involve anaerobic chamber (80% N₂, 10% H₂, 10% CO₂) at 37°C

• Methodological approach for studying atpE's role:

  • Compare ATP synthase activity and expression in mono-culture versus co-culture conditions

  • Evaluate atpE expression changes during different growth phases

  • Correlate ATP production with acetate generation and subsequent cross-feeding

To investigate the specific contribution of atpE to this metabolic relationship, researchers could employ genetic approaches such as creating conditional knockdowns of atpE to observe effects on acetate production and subsequent cross-feeding with E. rectale. This would help elucidate how ATP synthase function influences the ecological relationships within the gut microbiome.

What are the structural determinants of atpE that influence its self-assembly properties?

The self-assembly properties of ATP synthase subunit c (atpE) are primarily determined by specific structural features encoded in its primary sequence . Understanding these determinants is crucial for research on membrane protein assembly and function.

Key structural determinants:

• Transmembrane helices:

  • Recombinant ATP synthase subunit c contains two transmembrane α-helices connected by a polar loop

  • The helix-helix interactions are mediated by specific residues that facilitate oligomerization

  • The glycine-rich motifs (e.g., GIGTLGPGIGLGILFG in B. longum subsp. infantis atpE) promote tight packing of transmembrane segments

• Ion-binding site:

  • Contains a conserved carboxylic acid residue (typically aspartate or glutamate)

  • This residue is essential for proton translocation and influences the packing of subunits

• N-terminal and C-terminal regions:

  • The termini of atpE contribute to subunit-subunit interactions

  • Modifications to these regions (e.g., addition of tags) may affect self-assembly

Experimental evidence:
Studies with recombinant ATP synthase subunit c from E. coli have demonstrated that the protein self-assembles into ring structures in mild detergent solutions, even in the absence of other ATP synthase subunits . This indicates that the information required for c-ring formation is encoded within the primary structure of the protein itself.

Methodological approaches to study structure-assembly relationships:

• Site-directed mutagenesis:

  • Introduce specific mutations at key residues predicted to be involved in subunit-subunit interactions

  • Analyze the effects on oligomerization using methods described in FAQ 2.2

• Molecular dynamics simulations:

  • Model the dynamics of c-subunit interactions in a membrane environment

  • Predict the energetic contributions of specific residues to ring stability

• Hydrogen-deuterium exchange mass spectrometry:

  • Identify regions of the protein that are protected during oligomerization

  • Map the interaction surfaces between adjacent subunits

Understanding these structural determinants could inform the design of modified atpE proteins with enhanced stability or altered oligomerization properties for biotechnological applications.

How can researchers apply chemoproteomic approaches to study atpE reactivity and function?

Chemoproteomics offers powerful tools for studying the reactivity of specific amino acids in ATP synthase subunit c (atpE) and connecting these properties to protein function. Recent advances in chemoproteomic techniques can be applied to investigate atpE as follows:

Chemoproteomic approaches for atpE research:

• Isobaric Tag for Relative and Absolute Quantitation (isoTOP-ABPP):

  • This technique can identify highly reactive cysteines in the protein structure

  • Research has shown that highly reactive cysteines are often functionally important and enriched for genetic variants with high predicted pathogenicity

  • For atpE analysis, samples can be labeled with 10 or 100 μM iodoacetamide alkyne probe to identify reactive residues

• Amino acid reactivity profiling:

  • Beyond cysteines, other amino acids in atpE can be profiled for reactivity

  • This approach helps identify residues that may participate in proton translocation

  • Correlating amino acid reactivity with conservation across species can highlight functionally critical residues

• Integration with genetic variant data:

  • Mapping chemoproteomically-detected amino acids (CpDAAs) to genomic coordinates

  • Analyzing whether reactive residues in atpE correlate with genetic variants of high pathogenicity

  • This integrated approach can provide insights into structure-function relationships

Methodological considerations:

• Database selection and version control:

  • Careful attention to database update cycles and stable identifiers is essential for accurate mapping

  • Misidentification of labeled residues can occur due to database inconsistencies

• Experimental workflow:

  • Prepare membrane fractions containing recombinant atpE

  • Label with appropriate chemoproteomics probes

  • Analyze labeled peptides using LC-MS/MS

  • Map identified reactive residues to the protein structure

• Data analysis:

  • Use correlation analysis to compare reactivity profiles between different conditions

  • A Pearson correlation coefficient of 0.5 or higher indicates strong reliability of the data

By applying these chemoproteomic approaches, researchers can gain insights into which residues in atpE are particularly important for its function and stability, potentially identifying new targets for mutagenesis studies or drug development.

What are common challenges in expressing and purifying recombinant atpE and how can they be addressed?

Expressing and purifying membrane proteins like ATP synthase subunit c (atpE) presents several challenges. Here are common issues and evidence-based solutions:

Challenge 1: Low expression yields

• Causes: Toxicity to host cells, protein aggregation, inefficient translation

• Solutions:

  • Use tightly regulated expression systems (e.g., pET with T7 lysozyme co-expression)

  • Lower induction temperature (16-25°C) to slow protein synthesis and improve folding

  • Optimize codon usage for the expression host

  • Consider fusion partners that enhance solubility

Challenge 2: Inclusion body formation

• Causes: Rapid overexpression, improper folding, absence of chaperones

• Solutions:

  • Reduce inducer concentration (e.g., 0.1-0.5 mM IPTG instead of 1 mM)

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J)

  • Include membrane-mimetic environments during cell lysis

  • Alternatively, develop a refolding protocol from inclusion bodies

Challenge 3: Poor extraction from membranes

• Causes: Strong hydrophobic interactions, incomplete solubilization

• Solutions:

  • Screen detergents systematically (start with DDM, LDAO, or C12E8)

  • Use a combination of detergents and lipids

  • Optimize detergent-to-protein ratio

  • Include glycerol (10-20%) to stabilize the protein during extraction

Challenge 4: Oligomerization heterogeneity

• Causes: Detergent effects, concentration-dependent aggregation

• Solutions:

  • Maintain consistent protein concentration during purification

  • Use size exclusion chromatography to isolate homogeneous oligomeric states

  • Consider amphipols or nanodiscs for maintaining native-like environments

Purification optimization table:

For B. longum subsp. infantis atpE specifically, purification has been successful using His-tag affinity chromatography with final purity greater than 90% as determined by SDS-PAGE . The protein can be stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 to maintain stability.

How can researchers evaluate and improve the functional activity of recombinant atpE?

Evaluating and enhancing the functional activity of recombinant ATP synthase subunit c (atpE) requires systematic approaches focusing on assembly, stability, and proton translocation capability:

Activity assessment methods:

• Proton pumping assays:

  • Reconstitute purified atpE into liposomes containing pH-sensitive fluorescent dyes

  • Establish a pH gradient across the membrane and monitor its dissipation

  • Quantify proton flux rates under different conditions

  • Compare activity to established benchmarks for the native protein

• ATP synthesis coupling:

  • When reconstituted with other ATP synthase components, measure ATP production coupled to proton translocation

  • Use luciferase-based assays for real-time ATP detection

  • Evaluate the stoichiometry of protons translocated per ATP synthesized

• Structural integrity verification:

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

  • Thermal stability assays to determine protein melting temperature

  • Crosslinking studies to verify correct subunit arrangement in the c-ring

Activity optimization strategies:

• Lipid environment optimization:

  • Screen different lipid compositions to identify optimal membrane environment

  • Consider native B. longum membrane lipids for more physiologically relevant conditions

  • Test the effect of cardiolipin, which is known to associate with ATP synthases

• Buffer optimization:

  • Systematic variation of pH, ionic strength, and specific ions

  • Include osmolytes (e.g., glycerol, sucrose) to enhance stability

  • Test the effect of ATP and ADP on stability and assembly

• Protein engineering approaches:

  • Introduce disulfide bonds to stabilize the oligomeric structure

  • Remove flexible regions that might cause instability

  • Create chimeric proteins with known stable domains

Functional recovery table:

By systematically addressing these aspects, researchers can significantly improve the functional activity of recombinant atpE and ensure reliable results in their experiments.

How might genetic variants of atpE influence B. longum fitness in the gut microbiome?

The ATP synthase subunit c (atpE) is critical for energy production in Bifidobacterium longum, and genetic variations in this protein could significantly impact bacterial fitness in the competitive gut environment. Analysis of this question requires integrating evolutionary, structural, and ecological perspectives:

Functional implications of atpE variants:

• Energy harvesting efficiency:

  • Variants in the proton-binding site could alter the proton:ATP ratio

  • Changes in c-ring size (determined by subunit arrangement) might affect the thermodynamic efficiency of ATP synthesis

  • Such variations could provide adaptive advantages in different intestinal microenvironments

• Ecological niche specialization:

  • B. longum subsp. infantis is specialized for the infant gut, where it metabolizes human milk oligosaccharides

  • ATP synthase efficiency directly impacts its competitive ability in cross-feeding relationships

  • Variants optimized for specific pH ranges might enable colonization of different gut regions

• Evolutionary conservation analysis:

  • Highly reactive residues in proteins like atpE are often enriched for variants with high predicted pathogenicity scores

  • Comparative genomics across Bifidobacterium species could reveal selection pressures on atpE

Methodological approaches for investigation:

• Population genomics:

  • Sequence atpE from B. longum strains isolated from diverse human populations

  • Correlate variants with ecological factors (diet, geography, host age)

  • Apply selection pressure analysis to identify adaptively evolving sites

• Experimental evolution:

  • Culture B. longum under various conditions mimicking different gut environments

  • Sequence atpE after multiple generations to identify emerging variants

  • Functionally characterize these variants using approaches described in previous FAQ sections

• In vivo competition assays:

  • Introduce wild-type and variant B. longum strains into gnotobiotic mouse models

  • Track relative abundance over time to determine fitness advantages

  • Correlate with metabolomic analysis to link energy production efficiency to competitive outcomes

This research direction would provide valuable insights into how fundamental cellular components like ATP synthase evolve to optimize bacterial fitness in complex ecosystems like the human gut.

What potential applications exist for engineered atpE variants in probiotics or synthetic biology?

Engineered variants of ATP synthase subunit c (atpE) from Bifidobacterium longum subsp. infantis offer promising applications in probiotic development and synthetic biology. These applications leverage the central role of atpE in energy metabolism and cellular physiology:

Probiotic applications:

• Enhanced colonization ability:

  • Engineer atpE variants with improved proton translocation efficiency under acidic gut conditions

  • Optimize ATP production in the specific nutrient environment of the infant gut

  • Create strains with competitive advantages for establishing beneficial microbiota

• Controlled persistence:

  • Design atpE variants with conditional functionality (e.g., dependent on specific gut metabolites)

  • Develop probiotics with predictable gut transit times by modulating energy production capacity

  • Enable precise targeting of distinct gut regions through pH-dependent ATP synthase activity

• Therapeutic delivery systems:

  • Engineer B. longum with modified atpE to enhance survival during production and storage

  • Improve viability under gastric conditions to increase therapeutic delivery efficiency

  • Create energy-production switches that activate only in target tissues

Synthetic biology applications:

• Bioenergy production:

  • Engineer bacterial ATP synthases with modified c-subunits for enhanced ATP production

  • Develop biological systems for energy harvesting from proton gradients

  • Create hybrid energy-producing systems combining features from different bacterial species

• Biosensors:

  • Leverage the pH-sensitivity of atpE to develop whole-cell biosensors for environmental monitoring

  • Create reporter systems linked to ATP synthase activity

  • Develop diagnostic tools based on bacterial energy metabolism

• Minimal cell design:

  • Incorporate optimized atpE variants in minimal genome projects

  • Design synthetic cells with precisely controlled energy production capabilities

  • Study the fundamental requirements for life by manipulating this essential component

Methodological approaches for development:

• Directed evolution:

  • Apply selection pressure to libraries of atpE variants

  • Select for desired properties (stability, efficiency, pH optimum)

  • Characterize winners using functional assays described in previous sections

• Rational design:

  • Use structural information and molecular dynamics simulations to predict beneficial mutations

  • Focus on the proton-binding site and subunit interfaces

  • Apply insights from cross-species comparisons of ATP synthases

• High-throughput screening:

  • Develop assays linking cell growth or fluorescent reporters to ATP synthase function

  • Screen large libraries of variants in microfluidic systems

  • Validate promising candidates in more complex models

These applications represent the intersection of fundamental research on ATP synthase structure-function relationships with applied biotechnology, potentially leading to new therapeutic approaches and biotechnological tools.

What are the key considerations for researchers new to working with recombinant atpE?

For researchers beginning work with recombinant Bifidobacterium longum subsp. infantis ATP synthase subunit c (atpE), several key considerations should guide experimental design and implementation:

Critical experimental considerations:

• Expression system selection:

  • For structural studies, E. coli systems with high yield are suitable and can achieve >90% purity

  • For functional studies requiring post-translational modifications, consider yeast or baculovirus systems

  • Match the expression system to your specific research question and downstream applications

• Protein purification strategy:

  • Use affinity tags (His-tag) for efficient initial purification

  • Include membrane-mimetic environments throughout purification

  • Verify that the purification approach maintains the oligomeric state of atpE

• Functional validation:

  • Confirm proper folding and oligomerization before proceeding to functional studies

  • Establish appropriate positive and negative controls for activity assays

  • Consider the native biological context, including B. longum's role in cross-feeding interactions

• Storage and stability:

  • Maintain protein in appropriate buffer conditions (Tris/PBS with 6% Trehalose, pH 8.0)

  • Use glycerol (5-50%) for long-term storage at -20°C/-80°C

  • Aliquot samples to avoid repeated freeze-thaw cycles

• Data interpretation:

  • Consider the self-assembly properties of atpE when analyzing structural data

  • When mapping to genomic coordinates, be aware of database update cycles that can lead to misidentification of labeled residues

  • Compare results with existing literature on ATP synthase c subunits from related species

Recommended starting protocol:

• Express His-tagged recombinant atpE in E. coli using a pET-based vector system

• Extract membrane proteins using a mild detergent like DDM or LDAO

• Purify using Ni-NTA affinity chromatography followed by size exclusion

• Verify oligomeric state using BN-PAGE and/or SEC-MALS

• Reconstitute into liposomes for functional studies

• Store purified protein in aliquots containing 50% glycerol at -80°C

By carefully considering these aspects, researchers new to working with atpE can establish robust protocols and generate reliable data for advancing our understanding of this important component of bacterial energy metabolism.

How is research on B. longum atpE contributing to our broader understanding of bacterial ATP synthases?

Research on Bifidobacterium longum subsp. infantis ATP synthase subunit c (atpE) contributes significantly to our understanding of bacterial ATP synthases and microbial physiology in several key areas:

Evolutionary insights:

• B. longum is a key member of the infant gut microbiome with specialized metabolic capabilities

• Studying its ATP synthase provides insights into how this essential enzyme has adapted to the specific conditions of the gastrointestinal environment

• Comparative analysis with ATP synthases from other species helps elucidate the evolutionary constraints and adaptations of this fundamental biological machine

Structural biology advances:

• Research has confirmed that ATP synthase subunit c can self-assemble into ring structures even in the absence of other subunits

• This supports the concept that the ring-forming ability is encoded in the primary structure of the protein

• Understanding these self-assembly properties contributes to fundamental knowledge about membrane protein oligomerization

Microbial ecology and metabolism:

• B. longum participates in cross-feeding relationships with other gut bacteria, serving as an acetate producer

• ATP synthase function directly impacts these interactions by determining energy harvesting efficiency

• This research connects molecular mechanisms to ecosystem-level processes in the gut microbiome

Technological developments:

• Methods developed for expressing and characterizing B. longum atpE advance our capabilities for working with challenging membrane proteins

• Chemoproteomic approaches applied to ATP synthase components reveal reactive residues that may have functional significance

• Integration of protein-level data with genomic coordinates enhances our ability to predict the impact of genetic variations