Recombinant Gloeobacter violaceus UPF0060 membrane protein glr4174 (glr4174)

What is Gloeobacter violaceus UPF0060 membrane protein glr4174?

Gloeobacter violaceus UPF0060 membrane protein glr4174 is a 107-amino acid membrane protein (UniProt ID: Q7NDQ8) from the primitive cyanobacterium Gloeobacter violaceus. The full amino acid sequence is: MALLLFGLAAAAEIGGCFAFWSVLRLGKNPLWLAPGLVSLVVFAWLLTRSEATYAGRAYAAYGGVYIAASLVWLWLVEGTRPDRWDLAGALLCLAGAAVILFADRSP . This protein belongs to the UPF0060 family of membrane proteins, a group whose specific functions remain under investigation. The protein is particularly notable because it exists in a unique membrane organization without thylakoids, making it valuable for studying primitive photosynthetic membrane systems.

For research applications, recombinant versions of this protein are typically expressed with tags (commonly His-tags) to facilitate purification and characterization . The protein's hydrophobic nature and membrane localization present specific challenges for expression and functional studies, requiring specialized methodological approaches.

Why is Gloeobacter violaceus considered evolutionarily significant?

Gloeobacter violaceus holds exceptional evolutionary significance as it represents the most primitive extant cyanobacterium, occupying a basal position in phylogenetic analyses of organisms capable of plant-like photosynthesis . Multiple phylogenetic studies have confirmed its position as an early diverging lineage among cyanobacteria, chloroplasts, and photosynthetic eukaryotes . This placement makes proteins from this organism, including glr4174, invaluable for understanding the evolution of photosynthetic systems.

The evolutionary significance stems primarily from three key aspects:

• Complete absence of thylakoid membranes, a feature present in all other known cyanobacteria

• Unique structure of the photosynthetic apparatus

• Distinct arrangement of phycobilisomes and energy transfer pathways

These characteristics collectively suggest that Gloeobacter represents an evolutionary "living fossil," potentially providing insights into ancestral forms of photosynthesis . For researchers studying glr4174, this evolutionary context is essential for interpreting functional and structural data in a broader biological framework.

How is recombinant glr4174 protein typically expressed?

Recombinant glr4174 protein is typically expressed in E. coli expression systems with a histidine tag to facilitate purification . The expression methodology requires specific considerations due to the membrane-associated nature of the protein:

• Expression System Selection: The protein is commonly expressed in E. coli, but the strain selection is critical. BL21(DE3) or C41/C43(DE3) strains specifically designed for membrane protein expression often yield better results.

• Vector Design: Vectors containing an N-terminal His-tag are typically employed, as seen in the commercially available version (His-tagged, full length 1-107) .

• Induction Conditions: Lower induction temperatures (16-25°C) and reduced IPTG concentrations often improve the functional expression of membrane proteins by slowing production and allowing proper membrane insertion.

• Extraction Protocol: Gentle detergent extraction using mild non-ionic detergents (DDM, LDAO, or OG) helps maintain the native conformation during solubilization from membranes.

The expressed protein generally requires careful handling, including storage in buffers containing suitable detergents or reconstitution into membrane mimetics for functional studies .

How does the absence of thylakoids in Gloeobacter violaceus affect membrane protein organization including glr4174?

This unique organization creates several significant research considerations:

• Spatial Organization: The cytoplasmic membrane must organize both respiratory and photosynthetic electron transport chains, potentially creating unique protein-protein interactions unavailable in thylakoid-containing species.

• Lateral Heterogeneity: Studies suggest that the Gloeobacter cytoplasmic membrane contains specialized domains or rafts that functionally substitute for thylakoids, which may influence glr4174 localization and function.

• Protein Trafficking: Without thylakoid membranes, Gloeobacter lacks the complex protein trafficking systems found in other cyanobacteria, suggesting simpler insertion mechanisms for glr4174.

• Evolutionary Implications: The membrane organization potentially represents an ancestral state before the evolution of thylakoids, making glr4174 valuable for understanding early photosynthetic membrane evolution.

For researchers studying glr4174, this context necessitates considering potential associations with photosynthetic complexes and examining how the protein's function might relate to the unique membrane organization in Gloeobacter .

What experimental approaches are most effective for studying the function of glr4174?

Given the challenges associated with membrane proteins from primitive organisms, a multi-faceted experimental approach is most effective for studying glr4174 function:

• Comparative Genomics:

  • Identify homologs across cyanobacterial lineages

  • Map conservation patterns to infer functional regions

  • Perform phylogenetic profiling to identify potential interaction partners

• Structural Biology Approaches:

  • Cryo-electron microscopy of reconstituted protein

  • X-ray crystallography (challenging but potentially high-reward)

  • NMR spectroscopy of isotopically labeled protein in detergent micelles

  • Molecular dynamics simulations based on homology models

• Functional Characterization:

  • Liposome reconstitution assays to test transport functions

  • Patch-clamp electrophysiology if channel activity is suspected

  • Fluorescence-based assays to monitor potential conformational changes

  • Co-localization studies with known photosynthetic complexes

• Genetic Approaches:

  • Gene knockout or knockdown studies in Gloeobacter (challenging but informative)

  • Heterologous expression in other cyanobacteria to assess functional complementation

  • Site-directed mutagenesis of conserved residues followed by functional assays

• Proteomic Approaches:

  • Cross-linking mass spectrometry to identify interaction partners

  • Hydrogen-deuterium exchange to map solvent-accessible regions

  • Native mass spectrometry to determine oligomeric state

The integration of these approaches provides the most comprehensive understanding of glr4174 function, overcoming the limitations inherent to any single methodology.

How can site-directed mutagenesis of glr4174 be used to study its structure-function relationship?

Site-directed mutagenesis represents a powerful approach for exploring the structure-function relationship of glr4174. This methodology is particularly valuable given the challenges in obtaining high-resolution structural information for membrane proteins:

• Target Selection Strategy:

  • Conserved residues identified through multiple sequence alignments

  • Predicted functional motifs (e.g., the VILFADRSP C-terminal motif)

  • Charged residues within predicted transmembrane segments (potentially functionally significant)

  • The single cysteine residue at position 14, which may have structural or functional importance

• Methodological Workflow:

  • PCR-based mutagenesis using the recombinant expression vector as template

  • Confirmation of mutations by DNA sequencing

  • Expression and purification following established protocols for wild-type protein

  • Parallel characterization of wild-type and mutant proteins

• Functional Assays for Mutant Characterization:

  • Membrane integration efficiency (using protease protection assays)

  • Stability assessments (thermal shift assays, limited proteolysis)

  • Interaction studies with potential partners (pull-down assays)

  • Spectroscopic analysis to detect structural perturbations (CD, fluorescence)

• Experimental Design Considerations:

  • Include conservative and non-conservative substitutions

  • Create systematic alanine-scanning across predicted functional regions

  • Design cysteine-free variants for subsequent cysteine-scanning mutagenesis

  • Develop double-mutant cycles to test proposed interaction networks

This systematic mutagenesis approach allows researchers to map functional domains, identify critical residues, and develop testable hypotheses about glr4174's role in Gloeobacter's unique membrane system.

What expression systems yield the highest functional purity of recombinant glr4174?

Optimizing expression systems for recombinant glr4174 requires balancing yield with functional quality. Based on documented approaches for similar membrane proteins, the following systems can be considered:

• Bacterial Expression Systems:

  • E. coli C41/C43(DE3): Engineered specifically for membrane proteins, these strains contain mutations that prevent toxic effects of membrane protein overexpression .

  • E. coli Lemo21(DE3): Provides tunable expression through rhamnose-inducible regulation of T7 RNA polymerase activity.

  • E. coli with pBAD vectors: Arabinose-inducible system allows fine-tuning of expression levels.

• Cell-Free Expression Systems:

  • E. coli extract-based systems: Allow direct incorporation into nanodiscs or liposomes during synthesis.

  • Wheat germ extract systems: Potentially higher yield for difficult membrane proteins.

• Eukaryotic Expression Systems (for challenging cases):

  • Pichia pastoris: Yeast system capable of high-density cultures and post-translational modifications.

  • Insect cell (Sf9/Sf21): Baculovirus-based expression providing eukaryotic membrane environment.

The optimal expression conditions based on available research include:

For glr4174 specifically, the documented approach using E. coli with an N-terminal His-tag has proven successful for obtaining functional protein .

What purification protocols minimize denaturation of glr4174 membrane protein?

Purifying membrane proteins like glr4174 while maintaining their native conformation requires specialized protocols that differ significantly from those used for soluble proteins:

• Membrane Preparation and Solubilization:

  • Gentle cell disruption methods (sonication with cooling intervals or French press)

  • Isolation of membrane fraction by ultracentrifugation (100,000×g, 1h)

  • Solubilization using mild detergents (recommended: n-dodecyl-β-D-maltoside (DDM) at 1% w/v)

  • Extended solubilization period (4-16 hours) at 4°C with gentle agitation

• Affinity Purification:

  • Ni-NTA chromatography for His-tagged glr4174

  • Low imidazole (10-20 mM) in washing buffers to reduce non-specific binding

  • Gradient elution rather than step elution

  • Inclusion of detergent at concentrations above CMC throughout purification

• Buffer Optimization:

  • Maintenance of pH between 7.0-8.0 (Tris or phosphate buffers)

  • Inclusion of glycerol (10-20%) as a stabilizing agent

  • Addition of reducing agents if the cysteine residue is surface-exposed

  • Consideration of specific lipid additives that may stabilize the protein

• Post-Purification Handling:

  • Immediate buffer exchange to remove high imidazole

  • Concentration using specialized devices designed for detergent-containing samples

  • Storage at -80°C in buffer containing 6% trehalose for stability

  • Aliquoting to avoid freeze-thaw cycles

• Reconstitution Options:

  • Detergent dilution method for liposome incorporation

  • Nanodisc assembly for a more native-like membrane environment

  • Reconstitution in total lipid extract from Gloeobacter for maximum native context

Following purification, quality assessment through size-exclusion chromatography and negative-stain electron microscopy is recommended to confirm homogeneity and proper folding before proceeding to functional studies.

How should experimental conditions be adjusted when working with this primitive membrane protein?

Working with glr4174 requires specialized experimental adjustments due to its primitive evolutionary origin and membrane-associated nature:

• Buffer Considerations:

  • pH Range: Maintain pH between 7.0-8.0 to mimic the natural environment of Gloeobacter violaceus

  • Ionic Strength: Use moderate ionic strength (100-150 mM NaCl) to stabilize membrane proteins without inducing aggregation

  • Detergent Selection: Test multiple detergents (DDM, LDAO, OG) to identify optimal conditions for specific assays

  • Lipid Supplementation: Consider adding specific lipids (phosphatidylglycerol, cardiolipin) that may be required for function

• Temperature Considerations:

  • Storage Temperature: -80°C for long-term storage with 6% trehalose as a cryoprotectant

  • Working Temperature: 4-25°C for most assays to balance stability with physiological relevance

  • Thermal Sensitivity: Perform thermal stability assays to determine the functional temperature range

• Specialized Approaches for Functional Studies:

  • Reconstitution Systems: Consider nanodiscs or liposomes composed of cyanobacterial lipids

  • Native-like Environments: Bicelles or amphipols may provide better stability than detergent micelles

  • Light Conditions: If potential photosynthetic associations exist, conduct experiments under defined light conditions

• Analytical Method Adjustments:

  • Spectroscopic Methods: Correct for light scattering in detergent or liposome samples

  • Centrifugation Techniques: Adjust density gradients to account for detergent binding

  • Crystallization Screens: Use specialized membrane protein screens with lipidic cubic phase methods

• Stabilization Strategies:

  • Ligand Addition: Include potential metabolites or cofactors that might stabilize the protein

  • Engineering Approaches: Consider thermostabilizing mutations or fusion partners for challenging applications

  • Covalent Modification: Explore gentle crosslinking to stabilize oligomeric states if applicable

The reconstitution of lyophilized glr4174 requires particular attention, with recommendations to use deionized sterile water to a concentration of 0.1-1.0 mg/mL, followed by the addition of 5-50% glycerol as a stabilizing agent .

How can glr4174 contribute to understanding evolutionary transitions in photosynthetic membranes?

The glr4174 protein represents a unique opportunity to investigate evolutionary transitions in photosynthetic membrane organization due to its presence in Gloeobacter violaceus, the most primitive known cyanobacterium:

• Comparative Analysis Framework:

  • Phylogenetic Mapping: Compare glr4174 homologs across cyanobacterial lineages with different membrane complexities

  • Structural Conservation: Identify conserved motifs that persisted through evolutionary transitions

  • Functional Adaptation: Assess functional differences between glr4174 and homologs in thylakoid-containing species

• Methodological Approaches:

  • Ancestral Sequence Reconstruction: Computationally infer ancestral forms of glr4174

  • Heterologous Expression: Express ancestral and modern variants in model organisms

  • Functional Complementation: Test if glr4174 can functionally replace homologs in other species

• Evolutionary Hypotheses Testing:

  • Membrane Organization: Investigate if glr4174 interacts with primitive photosynthetic complexes

  • Functional Shifts: Determine if the protein's function changed during thylakoid evolution

  • Co-evolutionary Patterns: Identify correlated evolutionary changes with photosystem components

• Integration with Fossil Record:

  • Paleobiological Context: Relate findings to cyanobacterial fossil record dating back 2.7-3.5 billion years

  • Environmental Adaptations: Consider how early Earth conditions may have shaped membrane protein evolution

  • Transition Timing: Use molecular clock approaches to date potential functional transitions

Gloeobacter's status as a "living fossil" makes proteins like glr4174 invaluable for understanding how photosynthetic membranes evolved from simple cytoplasmic membrane-localized complexes to the sophisticated thylakoid systems found in modern cyanobacteria and chloroplasts .

What is the relationship between glr4174 and the unique photosynthetic apparatus of Gloeobacter?

The relationship between glr4174 and Gloeobacter's unique photosynthetic apparatus represents an intriguing research question that connects membrane protein biology with photosynthetic evolution:

• Structural Context:

  • In Gloeobacter, photosystems I and II, phycobilisomes, and electron transport components all reside in the cytoplasmic membrane rather than in specialized thylakoids

  • This creates a crowded membrane environment where glr4174 might have specific spatial relationships with photosynthetic complexes

• Potential Functional Hypotheses:

  • Spatial Organization: glr4174 might contribute to organizing photosynthetic complexes within membrane microdomains

  • Auxiliary Role: The protein could function as an accessory component in primitive energy transfer pathways

  • Regulatory Function: It might participate in redox regulation or sensing in the absence of thylakoid compartmentalization

• Experimental Investigation Approaches:

  • Co-localization Studies: Fluorescent tagging of glr4174 and photosystem components

  • Interaction Analysis: Co-immunoprecipitation or crosslinking mass spectrometry to identify binding partners

  • Physiological Measurements: Compare photosynthetic parameters in wild-type vs. glr4174-depleted strains

• Unique Features of Gloeobacter's Photosynthetic System:

  • Unusual photosystem I and II molecular structures with implications for membrane arrangement

  • Atypical phycobilisome organization with six peripheral phycocyanin/phycoerythrin rods bound to five horizontal allophycocyanin core rods

  • Distinctive energy transfer pathways that might involve novel membrane protein interactions

Understanding this relationship requires integrating membrane protein biochemistry with photosynthesis research, potentially revealing previously unrecognized connections between membrane organization and photosynthetic function in primitive systems.

What controls are essential when conducting localization studies of glr4174?

Membrane protein localization studies for glr4174 require rigorous controls to ensure reliable interpretation of results:

• Expression Level Controls:

  • Native Expression Baseline: Compare tagged protein expression to endogenous levels

  • Induction Gradient: Establish minimal expression levels that avoid artifacts from overexpression

  • Growth Phase Monitoring: Assess localization patterns across different bacterial growth phases

• Tagging Strategy Controls:

  • Tag Position Variants: Compare N-terminal vs. C-terminal tags to identify potential interference

  • Tag Size Comparison: Use both small epitope tags and fluorescent protein fusions to confirm patterns

  • Untagged Controls: Include parallel experiments with untagged protein using antibody detection

• Membrane Fractionation Controls:

  • Marker Proteins: Include known cytoplasmic, periplasmic, and membrane marker proteins

  • Multiple Fractionation Methods: Confirm results using different membrane isolation techniques

  • Protease Accessibility: Use protease protection assays to confirm topology predictions

• Imaging Controls:

  • Fixation Method Comparison: Compare live cell imaging with different fixation protocols

  • Autofluorescence Baseline: Establish Gloeobacter's natural fluorescence profile (significant due to photosynthetic pigments)

  • Resolution Controls: Include calibration standards appropriate for the imaging technique

• Functional Verification:

  • Activity Assays: Confirm that tagged constructs retain native functionality

  • Complementation Tests: Verify that tagged protein can rescue knockout phenotypes

  • Stability Assessment: Ensure that localization patterns aren't artifacts of protein degradation

These controls are particularly important given Gloeobacter's unusual membrane organization and the limited background knowledge about glr4174's native function and localization patterns .

How can researchers overcome challenges in structural characterization of glr4174?

Structural characterization of membrane proteins like glr4174 presents significant challenges that require specialized approaches:

• Sample Preparation Strategies:

  • Detergent Screening: Systematic testing of detergents for optimal protein stability

  • Lipid Cubic Phase (LCP): Utilizing LCP crystallization for membrane proteins resistant to traditional methods

  • Nanodiscs or SMALPs: Incorporating protein into nanodiscs or SMALPs (Styrene Maleic Acid Lipid Particles) for a more native-like environment

  • Fusion Partner Approach: Adding well-behaved protein domains (e.g., T4 lysozyme) to increase soluble surface area

• Crystallization Alternatives:

  • Single-Particle Cryo-EM: Bypassing crystallization requirements for proteins >50 kDa

  • Microcrystal Electron Diffraction: Using electron crystallography for very small crystals

  • NMR Approaches: Solution NMR for smaller membrane proteins or solid-state NMR for larger complexes

  • EPR Spectroscopy: Using site-directed spin labeling to determine distances between labeled sites

• Computational Approaches:

  • AlphaFold2 Prediction: Leveraging AI-based structure prediction tools

  • Molecular Dynamics Simulations: Refinement of predicted structures in membrane environments

  • Evolutionary Coupling Analysis: Using co-evolution signals to predict residue contacts

  • Hybrid Methods: Integrating low-resolution experimental data with computational models

• Functional Proxies for Structure:

  • Cysteine Scanning Accessibility: Mapping solvent-accessible surfaces

  • DEER Spectroscopy: Measuring specific distances between labeled sites

  • Hydrogen-Deuterium Exchange: Identifying protected regions of the protein

  • Cross-linking Mass Spectrometry: Detecting proximity relationships between residues

For glr4174 specifically, its relatively small size (107 amino acids) makes solution NMR a viable approach, particularly if expressed with isotope labeling (15N, 13C) in minimal media . Additionally, recent advances in cryo-EM for small membrane proteins may make this approach feasible when the protein is incorporated into larger scaffold systems like nanodiscs.

What are the most promising future research directions for glr4174?

Understanding the recombinant Gloeobacter violaceus UPF0060 membrane protein glr4174 offers several promising research avenues that connect evolutionary biology, membrane protein science, and photosynthesis research:

• Evolutionary Significance:

  • Comparative genomic analyses across cyanobacterial lineages to trace the evolutionary history of UPF0060 family proteins

  • Investigation of potential horizontal gene transfer events involving glr4174

  • Reconstruction of ancestral protein sequences to test hypotheses about primitive membrane systems

• Structural Biology Frontiers:

  • Application of emerging structural biology techniques to determine high-resolution structures

  • Integration of computational predictions with experimental validation approaches

  • Exploration of dynamic structural features through time-resolved methods

• Functional Characterization:

  • Development of genetic manipulation systems for Gloeobacter to enable in vivo studies

  • Investigation of potential roles in primitive photosynthetic membrane organization

  • Exploration of interactions with the unique photosystem arrangements in Gloeobacter

• Biotechnological Applications:

  • Evaluation of glr4174 as a potential model system for membrane protein engineering

  • Investigation of properties that might be valuable for synthetic biology applications

  • Development of glr4174-based experimental tools for studying membrane protein integration