Recombinant UPF0060 membrane protein HEAR0108 (HEAR0108)

Basic Research Questions

• What is UPF0060 membrane protein HEAR0108 and what is its significance in research?

  UPF0060 membrane protein HEAR0108 is a transmembrane protein from Herminiimonas arsenicoxydans with Uniprot accession number A4G1F7 . It belongs to the UPF0060 family of membrane proteins, which are conserved across various bacterial species. The significance of studying HEAR0108 lies in understanding membrane protein biology and potentially uncovering novel functions within bacterial membranes. While specific functions remain to be fully characterized, research on this protein contributes to the broader understanding of membrane protein structure-function relationships, which is critical given that membrane proteins constitute approximately 20-30% of gene coding proteins but remain underrepresented in structural databases .

• What are the optimal storage conditions for recombinant HEAR0108 protein?

  Recombinant HEAR0108 protein should be stored at -20°C for regular use, and at -20°C to -80°C for extended storage . The protein is typically provided in a Tris-based buffer with 50% glycerol that has been optimized for this specific protein. It's important to note that repeated freezing and thawing is not recommended as it can compromise protein integrity. For working with the protein, it's advised to create aliquots and store them at 4°C for up to one week to minimize freeze-thaw cycles . Some preparations recommend briefly centrifuging the vial before opening to ensure all material is at the bottom, especially if any product becomes entrapped in the cap during shipping and storage.

• What expression systems are commonly used for recombinant membrane proteins like HEAR0108?

  For membrane proteins like HEAR0108, several expression systems are employed, each with distinct advantages:

  Expression System	Advantages	Limitations	Suitable for
  E. coli	Cost-effective, rapid growth, high yields	Limited post-translational modifications, potential inclusion bodies	Small/medium membrane proteins, preliminary studies
  Yeast (P. pastoris, S. cerevisiae)	Eukaryotic post-translational modifications, higher yields than mammalian cells	Not all modifications match higher eukaryotes	More complex membrane proteins requiring some modifications
  Insect cells	Complex eukaryotic protein processing, good for larger proteins	More expensive, longer production time	Complex membrane proteins with multiple domains
  Mammalian cells	Native-like post-translational modifications	Most expensive, lower yields, longer production time	Mammalian membrane proteins requiring authentic modifications
  Cell-free expression	Avoids toxicity issues, direct incorporation into lipid environments	Lower yields, expensive	Toxic membrane proteins, rapid screening

  For HEAR0108 specifically, E. coli is frequently used as evidenced by product documentation, though the specific expression system may be determined during the production process based on yield and functionality requirements .

Advanced Research Questions

• What are the methodological challenges in structural characterization of UPF0060 family membrane proteins?

  Structural characterization of UPF0060 family membrane proteins faces several methodological challenges:

  • Extraction and Purification: Removing membrane proteins from their native lipid environment often disrupts their structure and function . For UPF0060 proteins, optimization of detergent type and concentration is critical for maintaining protein integrity.

  • Membrane Mimetics Selection: The choice between detergents, nanodiscs, SMALPs (styrene maleic acid lipid particles), or amphipols significantly impacts structural integrity . Each UPF0060 protein may require empirical testing to identify optimal conditions.

  • Crystallization Difficulties: Membrane proteins like those in the UPF0060 family are notoriously difficult to crystallize due to their hydrophobic surfaces and flexibility .

  • Cryo-EM Challenges: While increasingly powerful, single-particle cryo-EM still struggles with smaller membrane proteins like HEAR0108 (12 kDa range) .

  • NMR Constraints: Solution NMR requires stable protein-detergent complexes that maintain native-like conformations, which can be challenging to achieve with UPF0060 proteins.

  Methodological approaches to overcome these challenges include:

  1. Screening multiple detergents and lipid compositions systematically

  2. Employing mass photometry for rapid assessment of protein-detergent complex homogeneity

  3. Using in-drop dilution methods to minimize detergent interference in analytical techniques

  4. Considering fusion proteins or antibody fragments to increase protein size for cryo-EM studies

  5. Implementing computational prediction methods to guide experimental design

• How can I optimize purification protocols for HEAR0108 to maintain native conformation?

  Optimizing purification protocols for HEAR0108 requires careful consideration of several factors:

  1. Detergent Selection: Screen mild detergents like DDM, LMNG, or digitonin that preserve membrane protein structure. The optimal detergent should efficiently extract HEAR0108 while maintaining its structural integrity.

  2. Lipid Supplementation: Adding specific lipids during purification can stabilize membrane proteins. For bacterial membrane proteins like HEAR0108, phosphatidylethanolamine and cardiolipin may be beneficial.

  3. Buffer Optimization:

     • pH: Test a range (typically 6.5-8.0) to identify optimal stability

     • Salt concentration: Usually 150-300 mM NaCl, but may require optimization

     • Glycerol (10-20%): Enhances stability

     • Reducing agents: Include DTT or β-mercaptoethanol to prevent oxidation

  4. Purification Strategy:

     • Implement a two-step purification (e.g., affinity chromatography followed by size exclusion)

     • Maintain consistent detergent concentration above CMC throughout purification

     • Consider using nanodiscs or amphipols for the final preparation if functional studies are planned

  5. Quality Control:

     • Use mass photometry to assess homogeneity and oligomeric state

     • Conduct thermal stability assays (e.g., nanoDSF) to evaluate protein folding

     • Verify functionality through appropriate binding or activity assays

  6. Storage Conditions:

     • Store in buffer with 50% glycerol at -20°C for long-term storage

     • Avoid repeated freeze-thaw cycles

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

• What techniques are most effective for studying protein-lipid interactions of HEAR0108?

  Studying protein-lipid interactions of HEAR0108 requires specialized techniques:

  1. Native Mass Spectrometry:

     • Allows detection of specifically bound lipids

     • Can distinguish between tightly and loosely associated lipids

     • Requires careful optimization of ionization conditions to preserve non-covalent interactions

  2. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

     • Maps regions of the protein that are protected by lipid interactions

     • Can reveal dynamic changes in protein structure upon lipid binding

     • Requires specialized equipment and expertise

  3. Nanodiscs with Defined Lipid Composition:

     • Allows systematic testing of different lipid environments

     • Can be combined with functional assays to correlate lipid composition with activity

     • Particularly useful for identifying lipid specificity

  4. Fluorescence-Based Approaches:

     • Förster resonance energy transfer (FRET) between labeled protein and lipids

     • Microscale thermophoresis to measure binding affinities

     • Tryptophan fluorescence to monitor conformational changes

  5. Molecular Dynamics Simulations:

     • Predicts lipid binding sites and protein-lipid interactions

     • Can model the effect of different lipid environments on protein dynamics

     • Should be validated with experimental data

  Based on studies of similar membrane proteins, a combined approach using nanodiscs with defined lipid compositions followed by functional assays and structural studies provides the most comprehensive understanding of HEAR0108-lipid interactions .

• How can mass photometry be applied to characterize HEAR0108 and its interactions?

  Mass photometry offers significant advantages for characterizing HEAR0108:

  Methodology for HEAR0108 Characterization:

  1. Sample Preparation:

     • Prepare HEAR0108 in various detergents or membrane mimetics (0.01-0.1 mg/mL)

     • For detergent samples, use in-drop dilution method to minimize background signal

     • Include controls with known molecular weight standards

  2. Data Acquisition:

     • Apply 10-20 μL of sample to a clean glass coverslip

     • Record single-molecule interferometric scattering events

     • Collect data for 1-2 minutes (typically thousands of events)

  3. Analysis for HEAR0108:

     • Generate mass distribution histograms

     • Calculate molecular weights of observed species

     • Determine oligomeric state distribution

  4. Applications for HEAR0108:

     • Detergent Screening: Rapidly assess which detergents maintain HEAR0108 in a homogeneous state

     • Stability Assessment: Monitor changes in oligomeric state under various conditions

     • Protein-Protein Interactions: Detect binding of HEAR0108 to potential interaction partners

     • Incorporation into Nanodiscs: Verify successful incorporation and determine stoichiometry

  Advantages for HEAR0108 Studies:

  • Requires minimal sample (ng quantities)

  • Works directly in detergent solutions without complete detergent removal

  • Provides single-molecule resolution of heterogeneous populations

  • Enables rapid optimization of conditions (minutes per measurement)

  • Compatible with various membrane mimetics including detergents, amphipols, and nanodiscs

  Mass photometry has been successfully applied to membrane proteins with similar characteristics to HEAR0108, facilitating rapid optimization of purification conditions and assessment of sample quality for structural studies .

• What approaches can be used to investigate the potential functional relationship between HEAR0108 and other membrane proteins?

  Investigating functional relationships between HEAR0108 and other membrane proteins requires multi-faceted approaches:

  1. Co-immunoprecipitation and Pull-down Assays:

     • Generate antibodies or epitope-tagged versions of HEAR0108

     • Perform pull-downs to identify interaction partners

     • Validate interactions through reciprocal pull-downs

     • This approach successfully identified interactions between membrane proteins ACBD5 and VAPB

  2. Proximity Labeling:

     • Fuse HEAR0108 with BioID or APEX2 proximity labeling enzymes

     • Identify proteins in close proximity through biotinylation

     • Analyze biotinylated proteins via mass spectrometry

  3. Fluorescence-Based Interaction Assays:

     • FRET between fluorescently labeled HEAR0108 and potential partners

     • Bimolecular Fluorescence Complementation (BiFC)

     • Fluorescence correlation spectroscopy to detect complex formation

  4. Genetic Approaches:

     • Generate knockout or knockdown models of HEAR0108

     • Perform genetic screens to identify synthetic lethal or suppressor interactions

     • Analyze changes in cellular phenotypes

  5. Functional Reconstitution:

     • Co-reconstitute HEAR0108 with candidate partners in proteoliposomes

     • Measure functional parameters (e.g., transport activity, membrane permeability)

     • Test whether co-reconstitution affects functional properties

  6. Structural Studies of Complexes:

     • Use cross-linking mass spectrometry to identify interaction interfaces

     • Attempt co-crystallization or cryo-EM of complexes

     • Model interactions using computational approaches

  These approaches have been successfully applied to other membrane protein systems, such as the SAR1B-SURF4 interaction in lipoprotein transport and LRRC8B's role in calcium signaling , providing templates for investigating HEAR0108's functional partners.

• How can I design experiments to elucidate the potential role of HEAR0108 in membrane organization or cellular processes?

  Designing experiments to elucidate HEAR0108's role requires systematic investigation:

  1. Localization Studies:

     • Generate fluorescently tagged HEAR0108 constructs

     • Perform co-localization with organelle markers

     • Use super-resolution microscopy to examine membrane distribution patterns

     • Compare localization under different stress conditions

  2. Gene Expression Analysis:

     • Analyze expression patterns of HEAR0108 under various conditions

     • Identify co-regulated genes through transcriptomics

     • Determine if HEAR0108 expression correlates with specific cellular processes

  3. Loss-of-Function Studies:

     • Generate knockout or knockdown strains of H. arsenicoxydans

     • Perform comprehensive phenotypic analysis including:

       • Growth curves under various conditions

       • Membrane integrity assays

       • Stress response evaluations

       • Metabolomic profiling

     • Look for specific defects in membrane organization or function

  4. Gain-of-Function Studies:

     • Overexpress HEAR0108 in native or heterologous systems

     • Assess effects on membrane properties and cellular physiology

     • Determine if overexpression affects the localization or function of other membrane proteins

  5. Comparative Analysis with Other UPF0060 Proteins:

     • Compare phenotypes with those of other UPF0060 family members

     • Test for functional complementation between family members

     • Construct chimeric proteins to identify functional domains

     • This approach could leverage information from better-characterized family members like SAR2425

  6. Structural Impact on Membranes:

     • Reconstitute HEAR0108 in model membranes

     • Measure effects on membrane curvature, fluidity, or domain organization

     • Use techniques like atomic force microscopy to visualize membrane topology changes

  These experimental approaches parallel successful strategies used to discover functions of previously uncharacterized membrane proteins, such as the role of ACBD5-VAPB in peroxisome-ER associations and the identification of LRRC8B as an ER calcium leak channel .

Methodological Resources and Protocols

• What are the best practices for incorporating HEAR0108 into artificial membrane systems for functional studies?

  Incorporating HEAR0108 into artificial membrane systems requires careful consideration of methodological details:

  1. Proteoliposome Reconstitution:

     • Detergent-mediated reconstitution:

       • Solubilize lipids (typically E. coli polar lipids or defined mixtures) in detergent

       • Mix with purified HEAR0108 at protein:lipid ratios of 1:50 to 1:1000 (w/w)

       • Remove detergent using Bio-Beads or dialysis

       • Monitor reconstitution efficiency using density gradient centrifugation

     • Direct incorporation:

       • Add HEAR0108 during liposome formation

       • Use gentle methods like freeze-thaw cycles to improve incorporation

       • Particularly useful for proteins sensitive to detergents

  2. Nanodisc Assembly:

     • Mix purified HEAR0108, MSP (membrane scaffold protein), and lipids at optimized ratios

     • Initiate assembly by detergent removal

     • Purify assembled nanodiscs using size exclusion chromatography

     • Verify successful incorporation using mass photometry

     • This approach has been successfully used for other membrane proteins like the KcsA potassium channel

  3. Amphipol Trapping:

     • Purify HEAR0108 in detergent

     • Add amphipols (typically A8-35) at 1:5 protein:amphipol ratio

     • Remove detergent using Bio-Beads

     • Useful for maintaining protein stability for structural studies

  4. SMALP Formation:

     • Add SMA copolymer directly to membranes expressing HEAR0108

     • Extract native lipid environment surrounding the protein

     • Purify using affinity chromatography

     • Preserves native lipid interactions

  5. Functional Verification:

     • Confirm proper orientation using protease protection assays

     • Assess structural integrity using circular dichroism

     • Perform functional assays specific to the predicted role of HEAR0108

  These methods have been successfully applied to similar membrane proteins and can be adapted for HEAR0108 studies .

• What computational methods can help predict the structure and potential functions of HEAR0108?

  Several computational methods can provide insights into HEAR0108 structure and function:

  1. Homology Modeling:

     • Use structures of related UPF0060 family proteins as templates

     • Implement tools like MODELLER, SWISS-MODEL, or AlphaFold

     • Validate models using energy minimization and Ramachandran plot analysis

     • This approach was successful for modeling UCP2 structure based on ANT homology

  2. Ab Initio Modeling:

     • Leverage recent advances in protein structure prediction (AlphaFold2, RoseTTAFold)

     • Generate multiple models and assess confidence metrics

     • Focus analysis on high-confidence regions

  3. Molecular Dynamics Simulations:

     • Embed predicted structures in simulated lipid bilayers

     • Run extensive simulations (>100 ns) to assess stability and dynamics

     • Identify stable conformations and potential functional sites

     • Similar approaches revealed functional sites in UCP2

  4. Functional Site Prediction:

     • Use tools like ConSurf to identify evolutionarily conserved residues

     • Employ docking simulations to identify potential ligand binding sites

     • Apply machine learning approaches trained on known membrane protein functions

  5. Comparative Genomics:

     • Analyze genomic context of HEAR0108 across bacterial species

     • Identify co-occurring genes that suggest functional associations

     • Look for conservation patterns indicating important functional roles

  6. Integrative Approaches:

     • Combine multiple prediction methods

     • Weight predictions based on confidence scores

     • Integrate experimental data as it becomes available to refine models

  Successful application of these methods requires careful validation against experimental data and recognition of the limitations of each approach.

Human and Translational Research Relevance

• What is the current understanding of UPF0060 family proteins in bacterial physiology and potential relevance to human health?

  The UPF0060 family of membrane proteins remains largely uncharacterized, but emerging evidence suggests several important roles:

  1. Bacterial Membrane Organization:

     • Conserved across diverse bacterial species including pathogens

     • Sequence analysis suggests multiple transmembrane domains typical of transporters or channels

     • May contribute to membrane integrity and organization

  2. Potential Functional Roles:

     • Sequence similarity to known transporters suggests possible roles in:

       • Small molecule transport

       • Ion channel activity

       • Membrane signaling

     • Conserved motifs like "VYAAYGG" appear across family members (HEAR0108, SAR2425, Haur_1798)

  3. Pathogenic Relevance:

     • Present in several pathogenic bacteria including Staphylococcus aureus (SAR2425)

     • Could potentially contribute to:

       • Antibiotic resistance mechanisms

       • Adaptation to host environments

       • Stress responses during infection

  4. Human Health Implications:

     • As bacterial-specific membrane proteins, UPF0060 family members could represent:

       • Novel antibiotic targets

       • Diagnostic markers for specific bacterial infections

       • Opportunities for vaccine development

     • Understanding their structure and function could contribute to addressing antimicrobial resistance

  5. Research Status:

     • Currently at early research stages with limited functional characterization

     • Commercial availability of recombinant proteins like HEAR0108 facilitates further research

     • Structural studies using techniques like mass photometry may accelerate functional discoveries

  While direct human applications remain theoretical, bacterial membrane proteins like those in the UPF0060 family represent important targets for understanding bacterial physiology and developing new antimicrobial strategies.

• How can research on HEAR0108 contribute to broader understanding of membrane protein biology?

  Research on HEAR0108 can advance membrane protein biology in several significant ways:

  1. Methodological Advancements:

     • Serves as a model system for optimizing membrane protein purification and characterization

     • Contributes to refinement of techniques like mass photometry for membrane protein analysis

     • Provides opportunities to develop novel membrane mimetic systems

  2. Structural Biology Insights:

     • Adds to the limited structural data available for small membrane proteins

     • Helps identify common structural motifs and folding principles

     • Contributes to understanding how membrane proteins achieve stable conformations

  3. Evolutionary Perspectives:

     • Allows tracking of conserved features across diverse bacterial species

     • Illuminates how membrane proteins evolve while maintaining functional constraints

     • May reveal adaptations to specific environmental niches

  4. Functional Characterization:

     • Expands knowledge of membrane protein function beyond well-studied examples

     • Potentially uncovers novel membrane protein mechanisms

     • Helps connect sequence features to specific functions

  5. Membrane Biology Foundations:

     • Contributes to understanding how proteins and lipids interact in biological membranes

     • Provides insights into membrane organization principles

     • May reveal unknown aspects of membrane protein biogenesis

  6. Translational Applications:

     • Informs approaches for studying clinically relevant membrane proteins

     • Potential applications in synthetic biology for engineered membrane systems

     • May reveal druggable sites applicable to other membrane protein families

  By studying relatively simple bacterial membrane proteins like HEAR0108, researchers can establish fundamental principles that inform our understanding of more complex eukaryotic membrane protein systems, helping address the significant gap in membrane protein representation in structural databases .