Recombinant Alkaliphilus oremlandii UPF0316 protein Clos_0555 (Clos_0555)

What is Alkaliphilus oremlandii and why is it significant for research?

Alkaliphilus oremlandii strain OhILAs is a mesophilic, spore-forming, motile, gram-positive bacterium with low mole%GC content. It was initially isolated from Ohio River sediments using a basal medium with lactate and arsenate. Its significance stems from its unique physiological characteristics, particularly its ability to grow optimally at pH 8.4 and its respiratory capabilities. The organism can use arsenate and thiosulfate as terminal electron acceptors with various electron donors including acetate, pyruvate, formate, lactate, fumarate, glycerol, and fructose. This makes A. oremlandii particularly interesting for research into arsenic metabolism and detoxification mechanisms .

The organism's ability to transform both inorganic and organic arsenic compounds, including the organoarsenical 3-nitro-4-hydroxy benzene arsonic acid (roxarsone), suggests potential applications in bioremediation and environmental microbiology research. Its respiratory arsenate reductase, which is constitutively expressed, has been identified through biochemical and Western blot analyses, with the corresponding gene (arrA) being cloned and sequenced .

What is known about the UPF0316 protein family?

The UPF0316 protein family, to which Clos_0555 belongs, is classified as an "Uncharacterized Protein Family," indicating limited knowledge about its specific functions. UPF designations are assigned to protein families whose biological roles remain to be fully characterized. Based on sequence analysis, UPF0316 proteins typically contain conserved domains and structural features that suggest potential membrane-associated functions.

The amino acid sequence of Clos_0555 (MEALLGYLLIFVARLTDVSMATIRMIMVVKGKRVIAACIGFVEVSIYVVAIGKVLSGMDNPLNVLAYASGFATGNYVGIFLEEKMALGNIIAQVISDYEVEKLVRNVGFGVTVIEGGREGIRYILNVSLQRKHLSRLYQTVEEHDKKAFVTVTDARAIRGGYFAGMKK) shows characteristics consistent with membrane-associated proteins, including hydrophobic regions that could potentially form transmembrane domains .

How is the recombinant Clos_0555 protein typically prepared for research use?

The recombinant Clos_0555 protein is typically prepared using standard molecular biology and protein expression techniques. While specific production details may vary, the general methodology involves:

• Gene cloning: The gene encoding Clos_0555 (based on the UniProt accession A8mLK3) is amplified and inserted into an appropriate expression vector.

• Expression system selection: The choice of expression system depends on protein characteristics. For bacterial proteins like Clos_0555, E. coli is often the first choice, though other systems like yeast, insect cells, or mammalian cells may be considered if proper folding is an issue .

• Expression optimization: Conditions are optimized for maximum protein yield, including induction methods, temperature, and duration.

• Purification: The recombinant protein is typically purified using affinity chromatography (based on added tags), followed by additional purification steps as needed.

• Quality control: The final product undergoes testing for purity, integrity, and identity verification.

The commercially available recombinant protein is typically provided in a Tris-based buffer with 50% glycerol for stability and should be stored at -20°C, with extended storage at -80°C recommended .

What are the optimal conditions for handling and storing the recombinant Clos_0555 protein?

The optimal conditions for handling and storing recombinant Clos_0555 protein should follow these research-validated guidelines:

• Storage temperature: Store the protein at -20°C for regular use, or at -80°C for long-term storage to minimize degradation and maintain activity.

• Buffer composition: The protein is typically provided in a Tris-based buffer containing 50% glycerol, which helps maintain stability during freeze-thaw cycles .

• Aliquoting: To minimize freeze-thaw cycles, it is recommended to prepare small working aliquots upon receiving the protein. Working aliquots can be stored at 4°C for up to one week.

• Freeze-thaw cycles: Repeated freezing and thawing should be avoided as it can lead to protein denaturation and loss of activity .

• Handling during experiments: When working with the protein, maintain cold chain conditions whenever possible, and return unused portions to appropriate storage promptly.

• Documentation: Maintain detailed records of storage conditions, freeze-thaw cycles, and any observations regarding protein stability to ensure reproducibility across experiments.

What expression systems are most suitable for producing functional Clos_0555?

The selection of an appropriate expression system for producing functional Clos_0555 should be guided by considerations of protein characteristics and research requirements:

The amino acid sequence analysis of Clos_0555 suggests it may be a membrane-associated protein, which could present folding challenges in heterologous expression systems. For such proteins, specialized E. coli strains designed for membrane protein expression or yeast systems might provide optimal results . Codon optimization for the expression system of choice may also improve yields.

What purification strategies are most effective for Clos_0555?

Effective purification strategies for Clos_0555 should consider the protein's characteristics and potential challenges:

• Tag selection: The choice of affinity tag is crucial for efficient purification. Common options include:

  • His-tag: Allows for immobilized metal affinity chromatography (IMAC)

  • GST-tag: Enables glutathione affinity purification

  • MBP-tag: Can enhance solubility while providing affinity purification options

• Sequential purification approach:

  • Initial capture: Affinity chromatography based on the selected tag

  • Intermediate purification: Ion exchange chromatography to remove similarly charged contaminants

  • Polishing: Size exclusion chromatography for final purity

• Membrane protein considerations: If Clos_0555 exhibits strong membrane association characteristics, detergent selection becomes critical:

  • Screening different detergents for optimal solubilization

  • Considering mild detergents to maintain native conformation

  • Evaluating detergent removal strategies post-purification

• Quality control checks at each purification stage:

  • SDS-PAGE to assess purity

  • Western blotting for identity confirmation

  • Activity assays if functional characteristics are known

• Final formulation considerations:

  • Buffer optimization for stability

  • Addition of stabilizing agents like glycerol

  • Consideration of specialized storage conditions

The purification strategy should be optimized based on specific research requirements, balancing the need for high purity against yield and activity preservation.

How can structural studies be designed to characterize the Clos_0555 protein?

Designing structural studies for the characterization of Clos_0555 requires a methodical approach that combines computational and experimental techniques:

The selection of appropriate structural techniques should be guided by the specific research questions and the physicochemical properties of Clos_0555, with consideration for its potential membrane association and functional domains.

What functional assays can be developed to characterize the biological role of Clos_0555?

Developing functional assays for Clos_0555 requires a systematic approach to uncovering its biological role:

• Metabolic function exploration:

  • Given A. oremlandii's unique arsenate metabolism, investigate potential involvement in arsenic transformation pathways

  • Develop in vitro assays to test interaction with arsenate reductase components

  • Measure changes in arsenate/arsenite levels in the presence of purified Clos_0555

• Protein-protein interaction studies:

  • Implement pull-down assays using tagged Clos_0555 to identify interaction partners

  • Utilize yeast two-hybrid or bacterial two-hybrid systems for interaction screening

  • Confirm interactions through co-immunoprecipitation and crosslinking studies

  • Consider techniques like surface plasmon resonance for quantitative binding analysis

• Membrane association characterization:

  • Perform subcellular fractionation to determine localization

  • Use fluorescently labeled protein to visualize distribution in cell models

  • Implement liposome binding assays to assess membrane interaction properties

  • Investigate potential ion or small molecule transport functions using reconstituted systems

• Genetic approaches:

  • Generate knockout strains in A. oremlandii to observe phenotypic effects

  • Complement with wild-type and mutant variants to verify function

  • Utilize RNA-seq to identify genes co-regulated with Clos_0555

  • Implement CRISPR-Cas9 for precise genomic modifications

• Evolutionary analysis:

  • Identify homologs in related bacterial species

  • Perform comparative analysis of conserved residues

  • Map conservation patterns onto predicted structural models

  • Correlate evolutionary patterns with potential functional motifs

These approaches should be implemented iteratively, with results from initial studies informing the design of subsequent experiments to progressively build a comprehensive understanding of Clos_0555's biological role.

How might Clos_0555 be involved in A. oremlandii's unique arsenic metabolism?

The potential involvement of Clos_0555 in A. oremlandii's arsenic metabolism represents an intriguing research question that can be approached through multiple experimental strategies:

• Comparative genomic analysis:

  • Analyze the genomic context of Clos_0555 to identify proximity to known arsenic metabolism genes

  • Investigate co-occurrence patterns with arr and ars operons across bacterial species

  • Examine transcriptional regulation patterns in response to arsenic exposure

• Gene expression correlation studies:

  • Quantify Clos_0555 expression levels under varying arsenate/arsenite concentrations

  • Determine if expression changes correlate with known arsenic metabolism genes

  • Investigate potential co-regulation with the constitutively expressed arsenate reductase

• Protein-protein interaction mapping:

  • Test direct interactions between Clos_0555 and components of the respiratory arsenate reductase (ArrA, ArrB)

  • Investigate potential associations with proteins encoded by the ars operon

  • Perform crosslinking studies in vivo to capture transient interactions in native conditions

• Biochemical characterization:

  • Assess binding affinity of purified Clos_0555 to arsenate, arsenite, and organoarsenicals

  • Investigate potential enzymatic activities related to arsenic transformation

  • Evaluate structural changes in the protein upon arsenic compound binding

• Functional knockout studies:

  • Generate Clos_0555 deletion mutants and assess impact on:

    • Growth rates in arsenate-containing media

    • Arsenate reduction kinetics

    • Transformation of organoarsenicals like roxarsone

  • Complement with wild-type and site-directed mutants to confirm specific functional relationships

Given A. oremlandii's ability to couple the reduction of the nitro group of organoarsenicals to the oxidation of lactate or fructose in a dissimilatory manner , one hypothesis worth investigating is whether Clos_0555 plays a role in this unique metabolic pathway, potentially as a membrane component facilitating electron transfer or compound transport.

What are common challenges in expressing and purifying membrane-associated proteins like Clos_0555?

Membrane-associated proteins present unique challenges in expression and purification that require specialized strategies:

• Expression challenges:

  • Toxicity to host cells due to membrane disruption

  • Protein aggregation and inclusion body formation

  • Low expression yields compared to soluble proteins

  • Improper folding in heterologous expression systems

• Solubilization considerations:

  • Selection of appropriate detergents is critical and may require extensive screening

  • Detergent concentration must balance effective solubilization against protein destabilization

  • Some detergents may interfere with downstream applications

  • Native lipid environment may be required for proper folding and function

• Purification complications:

  • Detergent micelles can affect chromatographic behavior

  • Tag accessibility may be hindered by detergent micelles

  • Maintaining protein stability throughout multiple purification steps

  • Detergent exchange may be necessary for specific applications

• Methodological approaches to address these challenges:

  • Use specialized expression strains designed for membrane proteins

  • Consider cell-free expression systems

  • Implement fusion partners that enhance solubility

  • Optimize induction conditions (lower temperature, reduced inducer concentration)

  • Screen multiple detergents and stabilizing additives

  • Consider alternative membrane mimetics (nanodiscs, amphipols, SMALPs)

• Quality control considerations:

  • Assess protein homogeneity through size-exclusion chromatography

  • Verify proper folding using circular dichroism or fluorescence spectroscopy

  • Monitor detergent content using specialized assays

  • Validate functionality through binding or activity assays where possible

These challenges necessitate a systematic optimization approach, often requiring iterative refinement of conditions at each step of the expression and purification process.

How can researchers troubleshoot protein stability issues with Clos_0555?

Troubleshooting protein stability issues with Clos_0555 requires a methodical approach addressing multiple potential factors:

• Buffer optimization strategy:

  • Systematic screening of buffer components:

    • pH range testing (typically 6.5-8.5 for membrane proteins)

    • Salt concentration variations (50-500 mM)

    • Addition of stabilizing agents (glycerol, sucrose, arginine)

  • Thermal shift assays to quantitatively assess stability improvements

  • Dynamic light scattering to monitor aggregation propensity

• Storage condition optimization:

  • Compare stability at different temperatures (-80°C, -20°C, 4°C)

  • Assess impact of freeze-thaw cycles on activity and structural integrity

  • Evaluate lyophilization as a potential long-term storage solution

  • Consider addition of cryo-protectants for frozen storage

• Addressing oxidation sensitivity:

  • Add reducing agents if cysteine residues are present (DTT, β-mercaptoethanol)

  • Consider oxygen-free storage conditions

  • Evaluate impact of chelating agents to remove trace metals that catalyze oxidation

• Proteolytic degradation prevention:

  • Add protease inhibitors during purification and storage

  • Identify and mutate protease-susceptible sites if problematic

  • Remove flexible, protease-accessible regions through construct design

• Detergent considerations for membrane proteins:

  • Screen detergent types and concentrations for optimal stability

  • Consider detergent exchange to more stabilizing alternatives

  • Evaluate detergent-free alternatives like nanodiscs or amphipols

• Systematic stability assessment:

  • Implement regular quality control checkpoints using:

    • SDS-PAGE for degradation detection

    • Circular dichroism for secondary structure monitoring

    • Activity assays if available

    • Size-exclusion chromatography for aggregation assessment

Through methodical optimization of these parameters, researchers can identify conditions that maximize Clos_0555 stability for their specific experimental applications.

What strategies can be employed to investigate protein-protein interactions of uncharacterized proteins like Clos_0555?

Investigating protein-protein interactions of uncharacterized proteins requires a multi-faceted approach combining computational predictions with experimental validation:

• Computational interaction prediction:

  • Implement sequence-based predictive algorithms to identify potential interaction partners

  • Utilize structural models to identify potential interaction interfaces

  • Examine genomic context for gene proximity suggesting functional relationships

  • Analyze co-expression data to identify proteins with similar expression patterns

• Affinity-based experimental approaches:

  • Affinity purification mass spectrometry (AP-MS):

    • Express tagged Clos_0555 in native or heterologous systems

    • Perform pull-downs under varying conditions (detergent types, salt concentrations)

    • Identify binding partners through mass spectrometry

    • Implement appropriate controls to filter out non-specific interactions

  • Co-immunoprecipitation with candidate interactors:

    • Generate specific antibodies or use tag-based detection

    • Perform reciprocal co-IP experiments to confirm interactions

    • Test interactions under different physiological conditions

• Proximity-based methods:

  • BioID or TurboID approaches:

    • Generate fusion proteins with proximity labeling enzymes

    • Identify proteins in close proximity through biotinylation

    • Useful for capturing transient or weak interactions

  • FRET or BRET assays for direct interaction monitoring:

    • Generate fluorescent protein fusions

    • Measure energy transfer as evidence of direct interaction

    • Quantify interaction dynamics in real-time

• Genetic interaction methods:

  • Bacterial two-hybrid screening:

    • Particularly relevant for bacterial proteins like Clos_0555

    • Screen against genomic libraries from A. oremlandii

    • Validate hits through secondary assays

  • Synthetic genetic arrays to identify functional relationships:

    • Generate knockout strains and assess genetic interactions

    • Identify genes with synergistic or suppressive effects

• Validation and characterization:

  • Mutational analysis of interaction interfaces

  • Competition assays with peptides derived from interaction regions

  • In vitro reconstitution of protein complexes

  • Structural studies of identified complexes

These approaches should be applied in a strategic sequence, often beginning with computational predictions and broad screening methods, followed by targeted validation of specific interactions of interest.

How might research on Clos_0555 contribute to our understanding of arsenic bioremediation?

Research on Clos_0555 has potential implications for advancing arsenic bioremediation strategies through several mechanistic pathways:

• Enhanced understanding of bacterial arsenic metabolism:

  • If Clos_0555 is involved in A. oremlandii's arsenic transformation pathways, characterizing its function could reveal novel mechanisms for arsenic detoxification

  • Such knowledge could lead to the development of engineered bacterial strains with improved arsenic transformation capabilities

  • Understanding the protein's role may reveal rate-limiting steps in arsenic metabolism that could be targeted for enhancement

• Potential applications in biosensor development:

  • If Clos_0555 specifically interacts with arsenical compounds, it could be utilized as a recognition element in biosensors

  • Structure-function relationships could inform the design of protein-based detection systems

  • Engineered variants with enhanced specificity or sensitivity could improve current arsenic detection methods

• Contributions to bioremediation system design:

  • Understanding A. oremlandii's unique ability to transform both inorganic and organic arsenicals, including roxarsone , could inform bioreactor design

  • If Clos_0555 plays a role in the transport or metabolism of arsenicals, immobilized enzyme systems could be developed

  • Knowledge gained could facilitate the creation of synthetic microbial consortia optimized for arsenic removal from contaminated environments

• Evolutionary insights into arsenic resistance mechanisms:

  • Comparative analysis of Clos_0555 with homologs in other arsenic-metabolizing bacteria could reveal evolutionary adaptations

  • Such insights might identify convergent evolutionary strategies that could be mimicked in engineered systems

  • Understanding the co-evolution of different arsenic metabolism components could inform synthetic biology approaches

• Integration with existing bioremediation technologies:

  • Knowledge of molecular mechanisms could improve the efficiency of existing bioremediation approaches

  • Understanding protein-level functions could help optimize conditions for bacterial growth and activity in remediation settings

  • Insights could guide genetic modifications to enhance stability and performance in field applications

The research on Clos_0555 represents an important step in bridging molecular-level understanding with practical bioremediation applications, potentially contributing to more effective strategies for addressing arsenic contamination in environmental settings.

What comparative genomics approaches would be valuable for understanding the evolutionary context of Clos_0555?

Comparative genomics approaches provide valuable insights into the evolutionary context and functional significance of uncharacterized proteins like Clos_0555:

• Phylogenetic analysis of UPF0316 family proteins:

  • Construct comprehensive phylogenetic trees using homologs identified across bacterial species

  • Map the distribution of UPF0316 family proteins across taxonomic groups

  • Identify key evolutionary events such as gene duplications or horizontal gene transfers

  • Correlate evolutionary patterns with ecological niches and metabolic capabilities

• Synteny analysis:

  • Examine the genomic context of Clos_0555 homologs across multiple organisms

  • Identify conserved gene neighborhoods that suggest functional relationships

  • Map changes in genomic organization that may indicate evolutionary adaptation

  • Correlate synteny patterns with metabolic capabilities across species

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify regions under positive or purifying selection

  • Identify critical residues that have been conserved throughout evolution

  • Map selection patterns onto structural models to identify functional domains

  • Compare selection patterns with proteins of known function in arsenic metabolism

• Domain architecture comparison:

  • Analyze the presence of conserved domains or motifs across homologs

  • Identify lineage-specific insertions, deletions, or domain rearrangements

  • Correlate domain architecture changes with functional diversification

  • Reconstruct the evolutionary history of domain acquisitions

• Co-evolution network analysis:

  • Identify proteins that show correlated evolutionary patterns with Clos_0555

  • Build co-evolution networks to predict functional associations

  • Correlate co-evolution patterns with known metabolic pathways

  • Identify potential protein-protein interaction partners based on co-evolutionary signatures

These approaches should be implemented in concert to build a comprehensive evolutionary profile of Clos_0555, providing context for its potential functional role and guiding experimental investigations into its biological significance.

How can advanced structural biology techniques be applied to determine the function of Clos_0555?

Advanced structural biology techniques offer powerful approaches for elucidating the function of uncharacterized proteins like Clos_0555:

By integrating these advanced structural approaches with biochemical and genetic studies, researchers can develop detailed hypotheses about Clos_0555 function, guiding further experimental validation and ultimately contributing to a comprehensive understanding of its biological role.