Recombinant Hyphomonas neptunium ATP synthase subunit c (atpE)

What is Hyphomonas neptunium and why is its ATP synthase of research interest?

Hyphomonas neptunium is an alphaproteobacterium characterized by a unique budding mechanism where daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Originally described as Hyphomicrobium neptunium, it was isolated from the harbor of Barcelona, Spain . The organism has gained interest as a model for studying polar growth, asymmetric cell division, and bacterial development.

The ATP synthase of H. neptunium, particularly its subunit c (atpE), is of research interest because:

• It represents a membrane protein from a phylogenetically distinct bacterial group

• It functions in energy production within the stalk-bearing bacterium

• It has potential as a model for understanding membrane protein structure and function in budding bacteria

• It contributes to understanding evolutionary relationships between F-type ATPases across bacterial species

What is the molecular structure and composition of H. neptunium ATP synthase subunit c?

The H. neptunium ATP synthase subunit c (atpE) is a small membrane protein with the following characteristics:

• Amino acid sequence: MEGNITDGLKYVGAGLATLGMIGSALGVGNIFASFLDAAMRNPSAAPQQTGNLFIGMALA EALGIFSVLIAILILFVA

• Length: 78 amino acids (full length)

• UniProt ID: Q0C0X2

• Gene Name: atpE (also HNE_1920)

• Synonyms: ATP synthase F(0) sector subunit c, F-type ATPase subunit c, F-ATPase subunit c, Lipid-binding protein

• Predicted structure: Like other ATP synthase c subunits, it likely forms a ring structure within the F0 sector of the ATP synthase complex, consisting of primarily hydrophobic transmembrane helices

How is recombinant H. neptunium ATP synthase subunit c typically produced?

The recombinant H. neptunium ATP synthase subunit c is typically produced through heterologous expression in E. coli . The general methodology involves:

• Cloning the atpE gene into an expression vector with an N-terminal His-tag

• Transforming the construct into E. coli expression strains

• Inducing protein expression under optimized conditions

• Cell lysis and protein extraction (likely using detergents for this membrane protein)

• Purification via His-tag affinity chromatography

• Quality control assessment through SDS-PAGE (ensuring >90% purity)

• Lyophilization for storage and distribution

The recombinant protein can be reconstituted from lyophilized powder in deionized sterile water to achieve concentrations of 0.1-1.0 mg/mL, with recommended addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

How can H. neptunium ATP synthase subunit c be used for structural biology studies?

The structural biology of H. neptunium ATP synthase subunit c presents unique research opportunities due to its origin in a stalk-budding bacterium. Methodological approaches include:

Protein Preparation Protocol:

• Reconstitute the lyophilized protein in a buffer system optimized for structural studies (typically 20 mM Tris-HCl pH 8.0, 150 mM NaCl)

• For membrane protein studies, solubilize using mild detergents (e.g., DDM, LDAO, or amphipols)

• Perform size exclusion chromatography to ensure monodispersity and remove aggregates

• Concentrate to 5-10 mg/mL for crystallization trials

Structural Determination Methods:

• X-ray crystallography: Requires formation of well-ordered crystals, often challenging for membrane proteins

• Cryo-electron microscopy: Particularly useful for visualizing the c-ring assembly

• NMR spectroscopy: Suitable for studying dynamics and interactions in solution

• Molecular dynamics simulations: For predicting structural behavior based on the known amino acid sequence

Researchers should note that membrane proteins like atpE typically require specialized conditions for structural stability during purification and analysis.

What expression systems and genetic tools are available for studying H. neptunium proteins in their native context?

Recent methodological advances have enabled genetic manipulation of H. neptunium, allowing for native context studies:

Available Genetic Tools:

• Transformation protocol for H. neptunium via conjugation using E. coli WM3064 as a donor

• Selection markers: kanamycin (100/200 μg/ml) and rifampin (1/2 μg/ml) in liquid/solid medium

• Heavy metal-inducible promoters: copper and zinc responsive systems with low basal activity and high dynamic range

• Integrative plasmids with various fluorescent protein genes (EGFP, mCherry, etc.)

Experimental Protocol for H. neptunium Transformation:

• Harvest early-stationary-phase cultures of H. neptunium (2 ml) and plasmid-carrying E. coli WM3064 (1 ml)

• Wash pellets with Marine Broth (MB) medium

• Resuspend each in 100 μl medium containing 300 μM DAP

• Mix suspensions and spot onto an MB-agar plate with 300 μM DAP

• Incubate overnight at 28°C

• Scrape cells, wash twice in MB medium (without DAP)

• Resuspend in 1 ml MB medium

• Plate dilutions on selective MB-agar plates

• Incubate for 5 days at 28°C

These tools enable in vivo studies of atpE function, localization, and dynamics within the native H. neptunium cellular environment.

What are the optimal conditions for functional reconstitution of H. neptunium ATP synthase subunit c in liposomes?

For functional studies, reconstituting atpE into liposomes provides a system to study its bioenergetic properties. The following methodology is recommended:

Liposome Reconstitution Protocol:

• Prepare lipid mixture (typically 3:1 POPE:POPG) in chloroform

• Dry lipids under nitrogen and vacuum to form thin film

• Hydrate with buffer (10 mM HEPES, 100 mM KCl, pH 7.4)

• Subject to freeze-thaw cycles (5-10 times)

• Extrude through 400 nm polycarbonate filters

• Add detergent-solubilized atpE protein at lipid:protein ratio of 100:1

• Remove detergent using Bio-Beads or dialysis

• Collect proteoliposomes by ultracentrifugation

• Resuspend in assay buffer

Functional Assay Conditions:

• ATP synthesis: Monitor using luciferase-based ATP detection

• Proton transport: Measure using pH-sensitive fluorescent dyes (ACMA or pyranine)

• Membrane potential: Assess with potential-sensitive dyes (Oxonol VI)

These methods allow researchers to investigate the bioenergetic properties and proton translocation efficiency of the recombinant atpE protein.

How do I design experiments to study the effect of lipid environment on H. neptunium ATP synthase subunit c function?

Lipid environment significantly impacts membrane protein function. For investigating atpE:

Experimental Design Framework:

Methodological Approach:

• Prepare proteoliposomes with systematically varied lipid compositions

• Establish pH gradient (e.g., pH 4.5 outside, pH 7.5 inside)

• Add ADP and Pi to initiate ATP synthesis

• Quantify ATP production over time

• Correlate functional parameters with lipid environment variables

• Validate findings using molecular dynamics simulations

This systematic approach allows for determination of optimal lipid environments for atpE function and provides insights into how membrane composition may affect ATP synthase activity in the stalk structures of H. neptunium.

What are the most effective methods for studying protein-protein interactions involving H. neptunium ATP synthase subunit c?

Understanding how atpE interacts with other ATP synthase subunits and potential regulatory proteins requires specialized techniques:

Recommended Methods and Their Applications:

• Co-immunoprecipitation with tagged atpE

  • Express His-tagged atpE in H. neptunium using copper-inducible promoter system

  • Solubilize membranes with mild detergents

  • Capture complexes using Ni-NTA resin

  • Identify interaction partners by mass spectrometry

• Crosslinking Mass Spectrometry (XL-MS)

  • Apply membrane-permeable crosslinkers (DSS, BS3)

  • Digest crosslinked complexes

  • Analyze by LC-MS/MS

  • Map interaction interfaces computationally

• Förster Resonance Energy Transfer (FRET)

  • Generate fluorescent protein fusions (using available plasmids like pZGFPC-2)

  • Measure energy transfer between labeled proteins

  • Calculate proximity and orientation of interaction partners

• Split-GFP Complementation

  • Fuse protein partners with complementary GFP fragments

  • Express in H. neptunium using established transformation protocol

  • Visualize reconstituted fluorescence at interaction sites

• Bacterial Two-Hybrid System

  • Adapt for use in H. neptunium using copper/zinc inducible promoters

  • Screen for interactions using reporter gene expression

These methods can reveal the assembly process of the ATP synthase complex and identify novel regulatory interactions specific to H. neptunium.

How can I accurately assess the proton translocation efficiency of H. neptunium ATP synthase subunit c compared to homologs from other species?

Comparative analysis of proton translocation requires careful experimental design:

Standardized Comparison Protocol:

• Prepare matched proteoliposome systems:

  • Express and purify atpE from H. neptunium and comparative species (e.g., E. coli, C. crescentus)

  • Ensure identical protein:lipid ratios and liposome sizes

  • Verify incorporation using fluorescence correlation spectroscopy

• Establish proton gradient:

  • Load liposomes with pH-sensitive dye (ACMA or pyranine)

  • Create defined ΔpH using rapid dilution into buffer of different pH

• Measure proton flux:

  • Monitor fluorescence changes in real-time

  • Calculate initial rates of proton translocation

  • Normalize to protein concentration

• Data analysis and normalization:

This standardized approach allows for direct comparison of functional properties between atpE homologs and may reveal adaptations specific to the budding lifestyle of H. neptunium.

How does the membrane localization of ATP synthase in H. neptunium differ from other bacteria, and what role might this play in its unique budding division?

The unique cell biology of H. neptunium, with its stalk-mediated budding, raises intriguing questions about ATP synthase localization:

Investigative Approach:

• Generate fluorescently tagged atpE constructs using established plasmids (pZGFPN-2, pZCHYC-2)

• Transform H. neptunium using the conjugation protocol

• Induce expression with copper or zinc at defined cell cycle stages

• Perform high-resolution fluorescence microscopy to track localization

• Compare with membrane stains and cell cycle markers

Research Hypotheses to Test:

• ATP synthase may localize specifically to the stalk structure

• Differential distribution between mother cell and developing bud

• Dynamic reorganization during the cell cycle

• Potential role in energizing the stalk membrane

Understanding the spatial organization of ATP synthase could provide insights into how energy production is coordinated during the asymmetric division process of H. neptunium.

What are the molecular adaptations in H. neptunium ATP synthase subunit c that might relate to its stalk-budding lifestyle?

Comparative sequence and structural analysis may reveal adaptations specific to H. neptunium's unique lifestyle:

Analytical Framework:

• Perform multiple sequence alignment of atpE from diverse bacterial species

• Focus on H. neptunium, other stalked bacteria, and closely related non-stalked bacteria

• Identify conserved and divergent residues

• Map these onto predicted structural models

• Generate site-directed mutants to test functional significance

Key Adaptation Hypotheses:

• Modified proton-binding sites for efficiency in stalk environment

• Structural adaptations for curvature tolerance in narrow stalk

• Interaction surfaces for specialized regulatory proteins

• Lipid-binding regions adapted to stalk membrane composition

This approach can connect molecular features of atpE to the physiological requirements of H. neptunium's distinctive reproductive strategy.

How can high-throughput approaches be applied to study H. neptunium ATP synthase subunit c structure-function relationships?

Modern high-throughput methods can accelerate structure-function studies:

Deep Mutational Scanning Protocol:

• Generate comprehensive library of atpE single-point mutants

• Transform into H. neptunium using established methods

• Apply selection pressure (e.g., growth under ATP-limiting conditions)

• Use next-generation sequencing to identify enriched/depleted variants

• Map fitness effects to structural model

Complementary Approaches:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamic structural analysis

• Cryo-EM of ATP synthase complexes in different conformational states

• Computational molecular dynamics simulations of ion translocation

These approaches can generate comprehensive datasets on residue-level contributions to atpE function and assembly.

What are common challenges in working with recombinant H. neptunium ATP synthase subunit c and how can they be addressed?

Membrane proteins like atpE present specific challenges that require technical solutions:

Common Issues and Solutions:

Quality Control Checkpoints:

• Verify protein identity by mass spectrometry

• Assess purity by SDS-PAGE (>90%)

• Confirm proper folding using circular dichroism

• Validate function through proton translocation assays

These approaches can improve success rates when working with this challenging membrane protein.

How do I optimize the storage and handling of recombinant H. neptunium ATP synthase subunit c to maintain its functional integrity?

Proper storage and handling are critical for maintaining atpE functionality:

Storage Recommendations:

• Store at -20°C/-80°C upon receipt

• Aliquot to avoid repeated freeze-thaw cycles

• The lyophilized powder can be reconstituted to 0.1-1.0 mg/mL

• Add 5-50% glycerol to final concentration (50% is recommended)

• For working stocks, store aliquots at 4°C for up to one week

Handling Best Practices:

• Briefly centrifuge vials before opening

• Reconstitute in deionized sterile water

• Work with the protein in appropriate buffer systems (e.g., Tris/PBS-based buffer, pH 8.0)

• Minimize exposure to extreme temperatures or pH

• When possible, maintain in the presence of lipids or mild detergents

• For long-term storage of functional protein, consider storage in proteoliposomes

Following these guidelines will help maintain the structural and functional integrity of recombinant H. neptunium ATP synthase subunit c during experimental work.

What emerging technologies could advance our understanding of H. neptunium ATP synthase subunit c function in the context of bacterial cell biology?

Several cutting-edge approaches show promise for future research:

Emerging Technologies and Applications:

• Cryo-electron tomography

  • Visualize ATP synthase in situ within the stalk membrane

  • Map native distribution and organization without protein overexpression

• Single-molecule fluorescence microscopy

  • Track individual ATP synthase complexes during cell cycle

  • Measure rotational dynamics in living cells

• Microfluidics combined with time-lapse imaging

  • Study ATP synthase dynamics during budding process

  • Correlate with cell cycle markers and energy metabolism

• CRISPR-Cas9 genome editing

  • Adaptation of CRISPR tools for H. neptunium

  • Generate precise genomic modifications to atpE

  • Create conditional mutants for functional studies

• Synthetic biology approaches

  • Engineer minimal ATP synthase complexes

  • Study chimeric ATP synthases combining components from different species

These technologies can provide unprecedented insights into the role of ATP synthase in H. neptunium's unique bacterial cell biology.

How might comparative studies of ATP synthase subunit c across related alphaproteobacteria inform evolutionary adaptations to different lifestyles?

Evolutionary analysis can reveal adaptive changes in ATP synthase:

Comparative Research Framework:

• Collect atpE sequences from diverse alphaproteobacteria with different lifestyles:

  • Stalked budding bacteria (H. neptunium)

  • Stalked non-budding bacteria (C. crescentus)

  • Non-stalked relatives (Rhodobacterales)

• Perform phylogenetic analysis to identify:

  • Conserved functional residues

  • Lifestyle-specific adaptations

  • Convergent evolution events

• Test hypotheses with experimental approaches:

  • Heterologous expression of atpE variants

  • Functional complementation studies

  • Site-directed mutagenesis of key residues

This comparative approach can connect molecular evolution to ecological adaptations and provide insights into how ATP synthases have been modified throughout bacterial diversification.