Recombinant Bacillus licheniformis UPF0060 membrane protein BLi00854/BL03049 (BLi00854, BL03049)

What is the amino acid composition of Bacillus licheniformis UPF0060 membrane protein?

The UPF0060 membrane protein (BLi00854/BL03049) is composed of 108 amino acids with the following sequence: MMIAIGLFLLAGLAEIAGGYLVWLWLRESKPLWYGLAGGLTLIIYGVIPAFQAFPSFGRVYAAYGGVFIILAVLWGWLVDKKTPDLYDWAGAVICLQGVSVMLWAPRG . This sequence demonstrates characteristic features of membrane proteins, including hydrophobic stretches that likely form transmembrane domains. The protein is from the UPF0060 family, which belongs to uncharacterized protein families, indicating that while the sequence is known, detailed functional characterization remains incomplete. When analyzing this sequence, researchers should examine the hydrophobicity profile to predict the membrane-spanning regions and potentially exposed loops that might interact with substrates or other proteins.

How should researchers properly store and handle recombinant UPF0060 membrane protein to maintain its structural integrity?

Proper storage and handling of the recombinant UPF0060 membrane protein are crucial for maintaining its structural integrity and biological activity. The protein should be stored at -20°C for regular use, while long-term storage requires conservation at -20°C or -80°C . It is typically provided in a Tris-based buffer containing 50% glycerol, which has been optimized specifically for this protein's stability . Researchers should strictly avoid repeated freeze-thaw cycles as these can lead to protein denaturation and functional degradation. When working with the protein, it is recommended to prepare small working aliquots that can be stored at 4°C for up to one week to minimize freeze-thaw damage . For experiments requiring longer active periods, consider immobilization techniques that preserve native conformation or the use of stabilizing agents specific to membrane proteins.

How can single-molecule force spectroscopy (SMFS) be applied to study the structural dynamics of UPF0060 membrane protein in its native environment?

Single-molecule force spectroscopy (SMFS) represents a powerful approach to investigate the structural dynamics of membrane proteins like UPF0060 in their native environment. Researchers can adapt the methodology described by Galvanetto (2018), which involves isolating the plasma membrane of single cells to study membrane proteins in situ . For UPF0060 membrane protein, this would involve:

• Membrane Isolation: The "unroofing method" can be applied to B. licheniformis cells to isolate membrane fragments containing the native protein. This involves sandwiching cells between two glass plates and rapidly separating them to extract membrane portions .

• Force-Distance Curve Acquisition: Using atomic force microscopy (AFM), researchers can obtain force-distance (F-D) curves that represent the unfolding patterns of membrane proteins, including UPF0060 .

• Data Analysis: Implement unsupervised clustering procedures to detect sets of similar unfolding curves and compare them with those from purified proteins . The maximal contour length (L_cmax) histogram can encode the protein content and provide quantitative information about UPF0060 abundance in the membrane .

This methodology offers several advantages over traditional structural studies, including the ability to observe protein behavior at room temperature in the native membrane environment, where folding states may be influenced by physical-chemical properties of the cell membrane and interactions with nearby molecular partners .

What approaches can be used to investigate potential protein-protein interactions involving UPF0060 membrane protein in Bacillus licheniformis?

Investigating protein-protein interactions (PPIs) involving UPF0060 membrane protein requires specialized approaches due to the challenges associated with membrane protein biochemistry. The following methodological framework is recommended:

• In situ Cross-linking: Apply chemical cross-linkers to intact B. licheniformis cells followed by membrane isolation, protein extraction, and mass spectrometry analysis to identify proteins that are physically proximal to UPF0060 in the native membrane.

• Co-immunoprecipitation with Membrane-specific Modifications: Develop antibodies against UPF0060 or use tagged versions of the protein and employ detergent-based protocols optimized for membrane proteins to preserve interactions during extraction and precipitation.

• Split-reporter Systems: Implement bacterial two-hybrid systems or split-GFP approaches modified for membrane proteins to detect interactions in vivo.

• Native Membrane Isolation and Proteomic Analysis: Adapt the membrane isolation technique described for SMFS studies to preserve protein complexes, then analyze the composition using proteomics approaches . This method has shown promise for membrane proteomics at the single-cell level and could identify co-localized proteins that potentially interact with UPF0060 .

• Functional Correlation Studies: Design experiments to identify genes whose expression or function correlates with UPF0060 under various environmental conditions, potentially indicating functional relationships.

The choice between these approaches should consider the specific hypotheses about UPF0060's interactions and the available research infrastructure. Combining multiple methods would provide stronger evidence for genuine interactions.

How does the structure of UPF0060 membrane protein compare with other similar proteins across different bacterial species?

Comparative structural analysis of UPF0060 membrane protein across bacterial species requires a multi-faceted approach combining sequence analysis, structural prediction, and experimental validation:

Sequence Alignment and Conservation Analysis:

Structural Prediction and Analysis:

• Generate structural models using homology modeling and ab initio approaches

• Compare predicted structures to identify conserved folds and species-specific variations

• Analyze surface properties and potential functional sites

Experimental Validation:
Building on the SMFS methodology, researchers could perform comparative unfolding experiments on UPF0060 proteins from different species when reconstituted in lipid bilayers . The unfolding patterns (force-distance curves) would provide insights into structural similarities and differences that may not be apparent from sequence analysis alone. This approach has been successfully applied to other membrane proteins like Channelrhodopsin, Bacteriorhodopsin, and SthK .

The UPF0060 family's conservation pattern across species likely reflects both essential membrane functions and adaptations to specific ecological niches. B. licheniformis, as a soil bacterium with adaptations for surviving variable environmental conditions , may possess unique structural features in its membrane proteins compared to related species from different habitats.

What techniques can be employed to determine the functional role of UPF0060 membrane protein in Bacillus licheniformis?

Determining the functional role of UPF0060 membrane protein requires a comprehensive experimental strategy combining genetic, biochemical, and physiological approaches:

• Gene Knockout/Knockdown Studies:

  • Generate deletion mutants of BLi00854/BL03049 in B. licheniformis

  • Perform phenotypic characterization under various growth conditions

  • Assess changes in membrane properties, stress response, and metabolic activities

• Controlled Expression Systems:

  • Develop inducible expression constructs to modulate UPF0060 levels

  • Study dose-dependent effects on cellular physiology

  • Combine with transcriptomic and proteomic analyses to identify affected pathways

• Localization Studies:

  • Use fluorescent protein fusions or immunolocalization techniques

  • Determine subcellular distribution patterns under different conditions

  • Assess co-localization with other proteins of known function

• Transport/Channel Activity Assays:

  • Given its membrane localization, assess potential role in transport

  • Perform liposome reconstitution with purified protein

  • Measure transport of various substrates or ion channel activity

• Structural Biology Approaches:

  • Apply the SMFS techniques described earlier to compare unfolding patterns under different functional states

  • Correlate structural dynamics with potential functions

  • Identify structural elements critical for activity through site-directed mutagenesis

Each methodological approach should be designed with appropriate controls, considering B. licheniformis' growth requirements (optimal temperature around 50°C) and its transition between vegetative and spore states, which might affect membrane protein function .

How can researchers effectively distinguish between the roles of BLi00854 and BL03049 loci in experimental settings?

• Sequence and Annotation Verification:

  • Perform detailed bioinformatic analysis of both loci in the B. licheniformis genome

  • Confirm whether they represent identical, overlapping, or distinct genetic elements

  • Analyze the genomic context surrounding each locus for potential operonic structures

• Locus-Specific Genetic Manipulation:

  • Design precise genetic editing strategies targeting each locus individually

  • Create single and double mutants to assess independent and combinatorial effects

  • Complement mutants with each locus separately to confirm specific functions

• Transcriptional Analysis:

  • Develop locus-specific probes for qPCR or Northern blotting

  • Perform RNA-seq with specific analysis of each locus's expression pattern

  • Investigate potential differential expression under various conditions

• Protein Expression and Localization:

  • Generate antibodies or epitope tags specific to products of each locus

  • Determine if the proteins localize differently within the cell membrane

  • Assess potential complex formation between the products

• Functional Complementation Studies:

  • Express each locus in heterologous systems lacking similar genes

  • Test ability of each to rescue phenotypes associated with membrane function

  • Evaluate cross-complementation between the loci

This systematic approach would clarify whether BLi00854 and BL03049 represent truly distinct genetic elements with separate functions or are alternative designations for the same genetic unit.

What methods are recommended for analyzing post-translational modifications of the UPF0060 membrane protein?

Analyzing post-translational modifications (PTMs) of membrane proteins like UPF0060 presents unique challenges due to their hydrophobicity and membrane integration. The following methodological approach is recommended:

• Enrichment and Purification:

  • Use the recombinant protein expression system with carefully designed tags that don't interfere with modification sites

  • Develop membrane protein-specific extraction protocols that preserve PTMs

  • Consider native membrane isolation techniques to maintain the natural modification state

• Mass Spectrometry-Based Analysis:

  • Employ specialized proteomics workflows for membrane proteins

  • Use multiple proteases beyond trypsin to increase sequence coverage

  • Apply targeted MS approaches for suspected modification sites

  • Implement electron transfer dissociation (ETD) for PTMs that are labile under collision-induced dissociation

• Site-Specific Analysis Techniques:

  • Develop antibodies against common PTMs (phosphorylation, glycosylation, etc.)

  • Use chemical labeling strategies for specific modifications

  • Apply site-directed mutagenesis to confirm modification sites

• Functional Correlation Studies:

  • Compare modification patterns under different growth conditions

  • Correlate modifications with functional states of the protein

  • Use phosphatase/kinase inhibitors or glycosylation inhibitors to manipulate PTM status

• In situ Analysis:

  • Adapt the single-molecule force spectroscopy approach to detect structural changes associated with modifications

  • Compare unfolding patterns of modified vs. unmodified proteins to identify structural impacts of PTMs

For B. licheniformis UPF0060 membrane protein, researchers should consider the bacterium's natural growth conditions (optimal temperature around 50°C) when analyzing physiologically relevant modifications . Additionally, the protein's storage requirements (-20°C or -80°C) must be considered to preserve labile modifications during sample preparation .

How might UPF0060 membrane protein contribute to the ecological adaptations of Bacillus licheniformis in soil environments?

The UPF0060 membrane protein may play significant roles in B. licheniformis' adaptation to soil environments through several potential mechanisms:

• Membrane Integrity and Stress Response:
  As a membrane protein, UPF0060 might contribute to maintaining membrane integrity under the variable conditions found in soil environments. B. licheniformis demonstrates remarkable thermal tolerance (surviving well above its optimal growth temperature of 50°C) , suggesting specialized membrane adaptations in which UPF0060 could participate. The protein might help maintain appropriate membrane fluidity across temperature fluctuations or contribute to the transition between vegetative and spore states that allow B. licheniformis to survive harsh conditions .

• Nutrient Acquisition and Sensing:
  B. licheniformis is known for nutrient cycling capabilities and enzyme secretion . UPF0060 may function in nutrient sensing, transport, or signaling pathways that enable the bacterium to detect and utilize available resources in soil environments. This could be particularly relevant given the bacterium's role in breaking down complex biological materials, including feathers, through extracellular enzyme production .

• Interspecies Interactions:
  Soil environments host diverse microbial communities. UPF0060 might participate in recognition, communication, or competition with other microorganisms, potentially contributing to B. licheniformis' ecological fitness. The protein's structure and membrane location make it a candidate for involvement in processes that occur at the cell-environment interface.

To investigate these potential ecological roles, researchers should design experiments that examine UPF0060 expression and function under conditions that mimic soil environments, including variable temperature, pH, nutrient availability, and presence of competing microorganisms. Comparative studies with UPF0060 homologs from related Bacillus species occupying different ecological niches would provide additional insights into its adaptive significance.

What role might the UPF0060 membrane protein play in bacterial cell membrane organization and dynamics?

The UPF0060 membrane protein likely contributes to B. licheniformis membrane organization and dynamics through several potential mechanisms:

To study these potential roles, researchers could employ advanced microscopy techniques like super-resolution microscopy, fluorescence recovery after photobleaching (FRAP), and single-particle tracking to visualize and quantify membrane dynamics in living B. licheniformis cells with labeled UPF0060 protein.

How can computational approaches complement experimental studies of UPF0060 membrane protein structure and function?

Computational approaches offer powerful complementary tools for investigating UPF0060 membrane protein structure and function, particularly valuable given the experimental challenges associated with membrane proteins:

• Sequence-Based Analysis and Prediction:

  • Identify conserved motifs through multiple sequence alignments of UPF0060 family members

  • Predict functional sites using evolutionary conservation mapping

  • Employ machine learning algorithms trained on known membrane protein functions to predict UPF0060 function

  • Analyze the genomic context of BLi00854/BL03049 to identify potential functional relationships with neighboring genes

• Structural Modeling and Simulation:

  • Generate 3D structural models using homology modeling or ab initio approaches

  • Validate models against experimental data from SMFS unfolding patterns

  • Perform molecular dynamics simulations to study:

    • Protein behavior in membrane environments

    • Conformational changes under different conditions

    • Potential binding sites and interaction partners

• Systems Biology Integration:

  • Incorporate UPF0060 into genome-scale metabolic models of B. licheniformis

  • Predict system-wide effects of UPF0060 perturbation

  • Identify potential regulatory networks involving UPF0060

• Experimental Design Optimization:

  • Use computational predictions to guide targeted experimental investigations

  • Design optimal mutagenesis strategies to test structure-function hypotheses

  • Develop algorithms to analyze SMFS data more effectively, building on existing clustering approaches

• Data Integration Framework:

  • Integrate data from multiple experimental approaches:

    • SMFS unfolding patterns

    • Mass spectrometry proteomic data

    • Functional assay results

  • Develop visualization tools to represent complex membrane protein data

The computational approaches should be iteratively refined based on experimental validation, creating a feedback loop that progressively improves our understanding of UPF0060 structure and function. This integration is particularly valuable for UPF0060 as a member of an uncharacterized protein family, where computational predictions can guide efficient experimental exploration.

What are the primary challenges in purifying UPF0060 membrane protein while maintaining its native conformation, and how can they be addressed?

Purifying membrane proteins like UPF0060 while preserving native conformation presents several challenges, each requiring specific methodological solutions:

• Solubilization Challenges:

  • Challenge: Extracting membrane proteins from lipid bilayers without denaturation

  • Solution: Employ a gradient screening approach with different detergents (mild non-ionic detergents like DDM, LMNG, or digitonin) at various concentrations and conditions optimized for B. licheniformis membranes, which have adapted to function at temperatures around 50°C

• Stability During Purification:

  • Challenge: Maintaining structural integrity throughout multiple purification steps

  • Solution: Include stabilizing agents (glycerol, specific lipids) in all buffers as indicated in the storage recommendations for the recombinant protein (50% glycerol in Tris-based buffer) . Minimize time between purification steps and consider performing purification at temperatures closer to B. licheniformis' optimal growth conditions

• Functional Verification:

  • Challenge: Confirming that purified protein retains native functionality

  • Solution: Develop functional assays based on predicted roles; alternatively, use structural integrity assessments like circular dichroism or SMFS to compare unfolding patterns with those observed in native membranes

• Scale and Yield Issues:

  • Challenge: Obtaining sufficient quantities for structural and functional studies

  • Solution: Optimize expression systems specifically for membrane proteins, potentially using B. licheniformis itself as an expression host to provide the appropriate membrane environment and processing machinery

• Lipid Environment Reconstitution:

  • Challenge: Recreating appropriate lipid environment after purification

  • Solution: Use native membrane isolation techniques that preserve the protein in its original lipid environment , or reconstitute purified protein into liposomes with lipid compositions mimicking B. licheniformis membranes

• Aggregation Prevention:

  • Challenge: Preventing aggregation during concentration steps

  • Solution: Use amphipathic polymers, careful temperature control, and optimized buffer compositions; avoid repeated freeze-thaw cycles as specified in the handling recommendations

By systematically addressing these challenges through method optimization, researchers can significantly improve their chances of obtaining functionally intact UPF0060 membrane protein for detailed characterization.

How can researchers adapt single-molecule force spectroscopy protocols specifically for UPF0060 membrane protein studies?

Adapting single-molecule force spectroscopy (SMFS) protocols for UPF0060 membrane protein studies requires several specific methodological considerations:

• Sample Preparation Optimization:

  • Native Membrane Isolation: Adapt the "unroofing method" described in the literature specifically for B. licheniformis cells, considering their gram-positive cell wall structure which differs from the cells used in previous studies. Enzymatic pretreatment with lysozyme may improve membrane isolation efficiency.

  • Protein Density Control: Adjust isolation or reconstitution conditions to achieve appropriate UPF0060 density for single-molecule studies, avoiding overcrowding that would complicate individual protein identification.

• Force Probe Functionalization:

  • Specific Attachment Chemistry: Design custom chemistry for AFM tips that selectively interacts with exposed regions of UPF0060.

  • Tag-Based Approaches: Consider using the recombinant protein with appropriate tags positioned to facilitate controlled attachment without disrupting native structure.

• Measurement Parameters:

  • Force Range Calibration: Based on the amino acid sequence of UPF0060 , predict the likely mechanical stability and adjust force application rates accordingly.

  • Temperature Considerations: Conduct experiments at temperatures relevant to B. licheniformis biology (near 37°C for optimal enzyme function or 50°C for optimal growth) , which may differ from standard SMFS conditions.

• Data Analysis Adaptation:

  • Clustering Algorithm Refinement: Modify the unsupervised clustering approaches described in the literature to account for the specific expected unfolding patterns of UPF0060 based on its predicted structural elements.

  • Reference Pattern Establishment: Generate reference unfolding patterns using purified recombinant UPF0060 reconstituted in lipid bilayers, similar to the approaches used for other membrane proteins (Channelrhodopsin, Bacteriorhodopsin, and SthK) .

• Validation Strategy:

  • Identification Confirmation: Use the Bayesian meta-analysis approach described for identifying membrane proteins from SMFS data , incorporating information about UPF0060's sequence and predicted structure.

  • Mutational Analysis: Create specific mutations in predicted structural features of UPF0060 and observe changes in unfolding patterns to validate structural assignments.

By implementing these adaptations, researchers can generate reliable SMFS data specific to UPF0060, allowing for detailed characterization of its structural dynamics and potential functional states in conditions mimicking its native environment.

What considerations should be taken into account when designing expression systems for recombinant UPF0060 membrane protein production?

Designing expression systems for recombinant UPF0060 membrane protein production requires careful consideration of multiple factors to ensure high yield and proper folding:

• Host Selection Considerations:

  • Native vs. Heterologous Expression: Consider using B. licheniformis itself as an expression host to provide the native membrane environment and processing machinery

  • Alternative Bacterial Hosts: If heterologous expression is preferred, evaluate specialized strains of E. coli (C41/C43, Lemo21) designed for membrane protein expression

  • Cell-Free Systems: For difficult-to-express proteins, consider membrane-mimetic cell-free systems that avoid toxicity issues

• Vector and Promoter Design:

  • Induction Control: Implement tightly regulated promoter systems to prevent toxicity during growth phase

  • Expression Level Tuning: Design vectors allowing titration of expression levels to prevent overwhelming membrane insertion machinery

  • Fusion Partners: Consider fusions that enhance membrane targeting and folding while maintaining cleavability

• Growth and Induction Parameters:

  • Temperature Management: Account for B. licheniformis' optimal growth temperature (50°C) and optimal enzyme secretion temperature (37°C) when designing expression protocols

  • Membrane Biogenesis Support: Supplement with phospholipid precursors or specific lipids that support proper membrane protein folding

  • Induction Strategies: Employ slow induction approaches that allow time for proper membrane insertion and folding

• Extraction and Purification Strategy:

  • Tag Placement: Design constructs with tags positioned to avoid interference with membrane domains

  • Detergent Compatibility: Select solubilization and purification conditions compatible with downstream applications

  • Stability Considerations: Incorporate stabilizing agents (glycerol, specific lipids) throughout the purification process as indicated in handling recommendations

• Quality Control Metrics:

  • Folding Assessment: Implement methods to verify proper folding (e.g., circular dichroism, limited proteolysis)

  • Functional Validation: Develop activity assays based on predicted functions

  • Structural Integrity: Consider SMFS to compare unfolding patterns with native protein

These considerations should be systematically evaluated and optimized to develop a reliable production system for UPF0060 membrane protein that yields properly folded, functional protein suitable for structural and functional studies.