Recombinant Magnetospirillum magneticum UPF0060 membrane protein amb1014 (amb1014)

What are the structural characteristics of amb1014?

The amb1014 protein exhibits several key structural features characteristic of bacterial membrane proteins:

While high-resolution structural data (such as crystal structure or cryo-EM) is not yet available for amb1014, computational predictions suggest it adopts a structure typical of membrane proteins with alpha-helical transmembrane segments. Structural studies of membrane proteins typically require specialized techniques due to their hydrophobic nature and challenging expression and purification properties.

For detailed structural analysis, researchers should consider techniques like circular dichroism spectroscopy to analyze secondary structure content, or more advanced methods like NMR spectroscopy or cryo-electron microscopy when protein samples of sufficient purity and quantity are available .

How can amb1014 be expressed and purified for research?

Expression and purification of recombinant amb1014 can be achieved through the following methodological approach:

Expression System:
The protein can be successfully expressed in E. coli expression systems as demonstrated in the commercially available recombinant protein . The gene sequence should be codon-optimized for E. coli and cloned into an expression vector containing:

• An N-terminal His-tag for purification

• A strong inducible promoter (such as T7)

• Appropriate antibiotic resistance markers

Expression Protocol:

• Transform the expression construct into competent E. coli cells

• Inoculate transformed cells into LB medium containing appropriate antibiotics

• Grow cultures at 37°C until reaching an OD600 of 0.6-0.8

• Induce protein expression with IPTG (0.1-1.0 mM)

• Continue expression at a reduced temperature (16-25°C) for 16-20 hours to promote proper folding

Membrane Protein Extraction:

• Harvest cells by centrifugation at 4,000-6,000×g for 15 minutes

• Resuspend cell pellet in lysis buffer containing protease inhibitors

• Disrupt cells using sonication or a cell disruptor

• Remove cell debris by centrifugation at 10,000×g for 20 minutes

• Isolate membrane fraction by ultracentrifugation at 100,000×g for 1 hour

• Solubilize membrane proteins using appropriate detergents (e.g., n-dodecyl-β-D-maltoside, DDM)

Purification Strategy:

• Apply solubilized protein to Ni-NTA affinity chromatography

• Wash extensively to remove non-specifically bound proteins

• Elute with imidazole gradient (20-500 mM)

• Further purify by size exclusion chromatography

• Verify purity by SDS-PAGE and Western blotting

This approach typically yields purified recombinant amb1014 protein with purity greater than 90% as determined by SDS-PAGE, making it suitable for structural and functional studies .

What are the optimal storage conditions for recombinant amb1014?

Optimal storage conditions for recombinant amb1014 protein are critical to maintain its stability and functionality for research applications. Based on standard protocols for membrane proteins and the information available:

Short-term Storage (1 week):

• Store at 4°C in working aliquots

• Avoid repeated freeze-thaw cycles which can lead to protein denaturation

Long-term Storage:

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use

• Avoid repeated freeze-thaw cycles

Storage Buffer:

• Tris/PBS-based buffer, pH 8.0

• 6% Trehalose as a cryoprotectant

• Adding 5-50% of glycerol (final concentration) is recommended for long-term storage

Lyophilization and Reconstitution:

• The protein can be provided as a lyophilized powder

• Prior to use, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• After reconstitution, it's recommended to add glycerol (final concentration 50%) and aliquot for long-term storage at -20°C/-80°C

Stability Considerations:

• The vial should be briefly centrifuged prior to opening to bring contents to the bottom

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

These storage conditions help maintain the structural integrity and functional properties of the membrane protein for subsequent experimental applications.

How might amb1014 be involved in magnetotactic behavior?

While direct experimental evidence about amb1014's specific role in magnetotactic behavior is not provided in the available search results, we can draw inferences based on our understanding of magnetotactic bacteria and membrane proteins:

Magnetotactic bacteria like Magnetospirillum magneticum navigate using chains of magnetosomes (membrane-enclosed magnetic nanoparticles) that align with external magnetic fields. Similar proteins in M. magneticum, such as amb0994, have been studied and shown to play roles in magnetotaxis. For instance, amb0994 has been demonstrated to interact with MamK (a cytoskeletal protein involved in magnetosome chain arrangement) and influence the cellular response to magnetic field changes by affecting flagellar function .

As a membrane protein, amb1014 might contribute to magnetotactic behavior through several potential mechanisms:

• Magnetosome Membrane Organization: UPF0060 family proteins may contribute to the specialized membrane structure required for magnetosome formation.

• Signal Transduction: The protein might function in signaling pathways that respond to magnetic field changes, similar to the function described for amb0994 .

• Flagellar Motor Interaction: Like amb0994, amb1014 might influence flagellar rotation in response to magnetic torque.

Research approaches to study amb1014's role could include:

• Gene knockout using CRISPR-Cas9 genome editing techniques similar to those developed for M. magneticum AMB-1

• Protein localization studies to determine if amb1014 associates with magnetosome membranes

• Behavioral studies comparing wild-type and mutant strains' responses to magnetic field changes

These approaches would help establish whether amb1014 contributes to magnetotaxis and its specific function in this complex behavior.

What techniques are most effective for studying the structure-function relationship of amb1014?

Understanding the structure-function relationship of membrane proteins like amb1014 requires a multi-technique approach that addresses the unique challenges of these hydrophobic, often unstable proteins:

Structural Analysis Techniques:

Functional Analysis Approaches:

• Site-Directed Mutagenesis:

  • Systematically alter conserved residues

  • Express mutant proteins and assess effects on function

  • Target predicted transmembrane regions or conserved motifs

• Protein Reconstitution:

  • Incorporate purified amb1014 into liposomes or nanodiscs

  • Assess membrane integration and orientation

  • Study potential transport or signaling functions in controlled environments

• In vivo Complementation Studies:

  • Generate knockout strains using CRISPR-Cas9 as developed for M. magneticum AMB-1

  • Complement with wild-type and mutant versions

  • Quantify restoration of magnetotactic behavior

The Laboratory of Electron Transport Membrane Proteins and Structural Bioenergetics demonstrates that combining cryo-EM with other biochemical and biophysical methods is particularly effective for studying membrane proteins involved in energy transduction and cellular signaling . This integrated approach allows researchers to correlate structural features with specific functions, providing insights into the molecular mechanisms of protein activity.

How can CRISPR-Cas9 be used to study amb1014 function in vivo?

The successful application of CRISPR-Cas9 for genome editing in Magnetospirillum magneticum AMB-1, as described in the research literature, provides a powerful framework for studying amb1014 function . This methodological approach can be adapted specifically for amb1014:

CRISPR-Cas9 Applications for amb1014 Studies:

CRISPR-Cas9 Knockout Protocol for amb1014:

• sgRNA Design and Construction:

  • Identify target sequences within amb1014

  • Design sgRNAs with minimal off-target effects

  • Clone sgRNAs into a vector containing Cas9 expression cassette

  • Include homology arms flanking the amb1014 gene

• Delivery System:

  • Construct a conjugative plasmid containing the CRISPR-Cas9 system

  • Transfer into M. magneticum AMB-1 via conjugation with E. coli donor strain

  • Select transconjugants using appropriate antibiotics

• Mutant Selection and Verification:

  • Screen colonies using PCR to identify potential deletion mutants

  • Verify deletions by sequencing

  • Confirm absence of amb1014 expression by RT-PCR and Western blotting

CRISPR Interference Approach:

Research has demonstrated the successful development of a CRISPR interference system in M. magneticum AMB-1 by combining a nuclease-deficient Cas9 (dCas9) and single-guide RNA (sgRNA) . This system can be adapted to repress amb1014 expression:

• Design sgRNAs targeting the amb1014 promoter or early coding region

• Express dCas9 and the sgRNA in M. magneticum

• Quantify knockdown efficiency

• Analyze resulting phenotypic changes

Phenotypic Characterization:

Following gene modification, detailed phenotypic analysis should include:

• Magnetosome formation and arrangement (TEM imaging)

• Magnetic response behaviors (tracking cells under field reversals)

• Swimming behavior analysis

• Growth characteristics under various conditions

This comprehensive CRISPR-Cas9 approach, adapted from successful applications in M. magneticum AMB-1, provides a powerful toolkit for dissecting amb1014 function in vivo .

How might amb1014 interact with other proteins in magnetosome formation?

Understanding protein-protein interactions involving amb1014 in the magnetosome formation pathway requires systematic investigation using complementary techniques:

Potential Interaction Partners:

Based on knowledge of magnetosome formation, amb1014 may interact with:

• Other membrane proteins involved in magnetosome membrane invagination

• Proteins of the magnetosome membrane (Mam and Mms proteins)

• Cytoskeletal proteins like MamK that position magnetosomes

• Proteins involved in iron transport and biomineralization

Research on related proteins in M. magneticum has shown that some membrane proteins interact with cytoskeletal elements like MamK. For example, amb0994 has been demonstrated to interact with MamK, which plays a key role in magnetosome chain organization . Similar interactions might exist for amb1014.

Experimental Approaches for Interaction Mapping:

Validating Functional Relevance:

• Mutational Analysis:

  • Create point mutations at predicted interface residues

  • Assess effects on interaction strength

  • Evaluate phenotypic consequences in vivo

• Co-expression Analysis:

  • Analyze transcriptional co-regulation with known magnetosome genes

  • Identify conditions that alter expression patterns

By systematically mapping the interaction network of amb1014, researchers can place this protein within the broader context of the magnetosome formation pathway and better understand its functional contributions to magnetotactic behavior, potentially revealing similar roles to those established for amb0994 .

What is the role of amb1014 in electron transport and energy metabolism?

While specific information about amb1014's role in electron transport is not directly provided in the search results, analyzing its potential functions as a membrane protein in magnetotactic bacteria provides valuable research directions:

Magnetospirillum magneticum AMB-1 is an alpha-proteobacterium with microaerophilic respiratory metabolism that must balance energy generation with magnetosome formation. The laboratory of Electron Transport Membrane Proteins researches membrane proteins essential for energy transduction and cellular defense/signaling . These proteins typically transport electrons from high-energy donors to lower energy acceptors, coupled to proton pumping across membranes .

As a membrane protein, amb1014 could potentially participate in:

• Electron Transport Chain Components: It may function as an uncharacterized component of the respiratory chain, potentially involved in electron transfer to or from magnetosomes.

• Redox Sensing: It could serve as a redox sensor linking environmental conditions to magnetosome formation.

• Proton Transport: It might participate in proton translocation associated with energy conservation.

Experimental Approaches to Investigate Energy Metabolism Roles:

The electron transport chain complexes generally fall into five groups: complex I (NADH-coenzyme Q oxidoreductases), complex II (non-proton-pumping coenzyme Q reductases), complex III (coenzyme Q-cytochrome c oxidoreductases), complex IV (oxidases), and complex V (ATP synthase) . Determining which of these systems amb1014 might be associated with would provide insight into its specific role in energy transduction.

How can imaging techniques be adapted to study amb1014 trafficking and localization?

Recent advances in real-time imaging of axonal membrane protein life cycles provide valuable methodological approaches that can be adapted to study amb1014 trafficking and localization in magnetotactic bacteria :

Protein Tagging and Labeling Strategies:

Researchers can engineer amb1014 with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability . This approach provides flexibility for investigating the trafficking and spatiotemporal regulation of membrane proteins in different cellular compartments.

Key Imaging Procedures Adaptable to amb1014 Studies:

• Protein Transport Visualization:

  • Monitor anterograde and retrograde transport of tagged amb1014

  • Track protein movement relative to magnetosome chains

  • Analyze transport kinetics and directionality

• Multi-protein Tracking:

  • Simultaneously label amb1014 and other magnetosome proteins with different fluorophores

  • Study co-transport dynamics and potential protein-protein interactions in vivo

  • Determine temporal sequence of protein localization during magnetosome formation

• Membrane Dynamics:

  • Investigate exocytosis and endocytosis of amb1014

  • Monitor protein insertion into magnetosome membranes

  • Study protein recycling and turnover rates

• Subcellular Localization:

  • Determine precise localization relative to magnetosome chains

  • Analyze protein distribution in response to magnetic field changes

  • Correlate localization patterns with cellular functions

Technical Implementation:

Using microfluidic chambers similar to those described for neuronal studies , researchers can physically compartmentalize bacterial cells to analyze protein movement between different cellular regions. Additionally, the open-source software developed for high-throughput analysis of imaging data can be adapted for bacterial studies .

This comprehensive imaging approach would enable researchers to study the dynamics of amb1014 membrane protein homeostasis, addressing fundamental questions about its trafficking, localization, and potential role in magnetosome formation or other cellular processes in magnetotactic bacteria.