Recombinant Methylobacterium nodulans UPF0060 membrane protein Mnod_6500 (Mnod_6500)

What is Methylobacterium nodulans UPF0060 membrane protein Mnod_6500?

Mnod_6500 is a full-length membrane protein (107 amino acids) belonging to the UPF0060 protein family found in Methylobacterium nodulans, a unique bacterial species capable of inducing nitrogen-fixing root nodules in specific legume plants of the Crotalaria genus . This protein is part of a bacterial system that has both methylotrophic metabolism and nitrogen fixation capabilities, making it of particular interest for understanding membrane protein function in specialized bacterial symbionts . The recombinant version typically includes an N-terminal His-tag and is expressed in E. coli expression systems for research purposes . The complete amino acid sequence is: MTTLLAYVGAALAEIAGCFAVWAWLRLGRSPLWLGPGLASLALFAVLLTRVESGAAGRAYAAYGGVYIAASLVWLWGVEGQRPDRWDLGGAALCLAGTAVILLGPRG .

How should Recombinant Mnod_6500 protein be stored and reconstituted for experimental use?

For optimal preservation and experimental reliability, Mnod_6500 protein should be stored at -20°C to -80°C upon receipt, with aliquoting recommended to avoid repeated freeze-thaw cycles that can compromise protein integrity . The protein is typically supplied as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 .

For reconstitution, follow this protocol:

• Briefly centrifuge the vial before opening to ensure all material is at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is standard) for long-term storage

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store working aliquots at 4°C for up to one week for ongoing experiments

This methodological approach ensures maximum stability and activity retention for sensitive membrane protein specimens.

What expression systems are most effective for producing functional Mnod_6500 protein?

E. coli expression systems are the standard choice for recombinant production of Mnod_6500 protein as demonstrated in commercial preparations . The effectiveness of this system likely stems from several factors that should be considered when designing expression protocols:

• Codon optimization: Since Methylobacterium nodulans has different codon usage than E. coli, codon optimization of the synthetic gene may be necessary to ensure efficient translation.

• Membrane protein expression challenges: As with many membrane proteins, expression can be hampered by toxicity, protein misfolding, or inclusion body formation . Using specialized E. coli strains designed for membrane protein expression (such as C41/C43 or Lemo21) may improve yields.

• Induction conditions: Lower induction temperatures (16-25°C) and reduced inducer concentrations often improve the folding and membrane insertion of membrane proteins.

• Extraction considerations: Efficient membrane extraction using appropriate detergents is critical for maintaining the native structure of membrane proteins.

The effectiveness of alternative expression systems such as yeast, insect cells, or cell-free systems has not been specifically documented for Mnod_6500, but these could be explored for applications requiring different post-translational modifications or higher yields of properly folded protein.

What structural characterization methods are most suitable for Mnod_6500 membrane protein analysis?

Structural characterization of Mnod_6500 requires specialized approaches due to its membrane-embedded nature. A multi-technique approach is recommended:

• X-ray Crystallography: Challenge with membrane proteins like Mnod_6500 lies in generating well-diffracting crystals. This typically requires:

  • Detergent screening to identify optimal solubilization conditions

  • Lipidic cubic phase crystallization methods

  • Protein engineering to improve crystallization properties

• Cryo-Electron Microscopy (Cryo-EM): Particularly valuable for membrane proteins that resist crystallization:

  • Sample preparation in nanodiscs or amphipols can preserve native-like lipid environment

  • Single-particle analysis for structure determination

  • Subtomogram averaging for in situ structural studies

• Nuclear Magnetic Resonance (NMR): For dynamic structural information:

  • Solution NMR for smaller membrane proteins or fragments

  • Solid-state NMR for membrane-embedded proteins in native-like lipid bilayers

• Small-Angle X-ray Scattering (SAXS): For low-resolution envelope structures and conformational ensembles in solution

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): For protein dynamics and solvent accessibility mapping

Given the relatively small size of Mnod_6500 (107 amino acids), a combination of solution NMR and complementary techniques like HDX-MS would likely provide the most comprehensive structural information . For integration with membrane protein design approaches, computational modeling based on the DeGrado Lab's membrane protein design frameworks could also be informative for predicting structural features .

How can protein-protein interactions involving Mnod_6500 be reliably detected and validated?

Detecting and validating protein-protein interactions (PPIs) for membrane proteins like Mnod_6500 presents unique challenges. A robust approach combines multiple complementary methods to minimize false positives and negatives:

Primary Detection Methods:

• Modified Yeast Two-Hybrid Systems:

  • Split-ubiquitin membrane yeast two-hybrid (MYTH) system adapted for membrane proteins

  • Consider limitations as interaction must occur in nucleus, which may affect membrane protein behavior

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Use His-tag for pulldown assays followed by MS identification of binding partners

  • Employ crosslinking to capture transient interactions

  • Include proper detergent controls to distinguish true interactions from non-specific membrane protein aggregation

• Proximity-Based Labeling:

  • BioID or APEX2 fusion proteins for in vivo proximity labeling

  • Particularly valuable for identifying transient or weak interactions in native membrane environments

Validation Methods:

• Fluorescence Resonance Energy Transfer (FRET):

  • For studying interactions in live cells

  • Can provide spatial and temporal resolution of interactions

• Bimolecular Fluorescence Complementation (BiFC):

  • Direct visualization of protein interactions in cells

  • Consider irreversibility of the fluorescent complex formation

• Surface Plasmon Resonance (SPR) or Microscale Thermophoresis (MST):

  • For quantitative binding parameters (Kd values)

  • Requires careful preparation of membrane protein samples in suitable detergents or nanodiscs

Integration and Scoring:

Research by von Mering et al. shows that using the intersection of multiple detection methods dramatically reduces false positives but at the cost of coverage . To optimize both specificity and sensitivity, implement a confidence scoring system integrating:

The integration of computational prediction methods with experimental validation has been shown to improve reliability in protein interaction studies .

What functional roles might Mnod_6500 play in Methylobacterium nodulans biology given its membrane localization?

Based on contextual analysis of Methylobacterium nodulans biology and the characteristics of UPF0060 family membrane proteins, Mnod_6500 likely contributes to one or more of these functional areas:

• Symbiotic Nitrogen Fixation Processes:
  Methylobacterium nodulans is unique among methylotrophs in its ability to form nitrogen-fixing nodules with Crotalaria species . Mnod_6500 could potentially:

  • Facilitate metabolite exchange between bacteria and host plant

  • Participate in signaling cascades regulating nodulation

  • Contribute to creating microaerobic conditions necessary for nitrogenase activity

• C1 Metabolism:
  As a methylotrophic bacterium, M. nodulans grows on C1 compounds like methanol, formate, and formaldehyde . Mnod_6500 might:

  • Transport C1 compounds across the membrane

  • Participate in sensory mechanisms for detecting environmental C1 compounds

  • Help maintain membrane integrity under metabolic stress conditions

• Environmental Adaptation:
  The membrane localization suggests potential roles in:

  • pH homeostasis or stress response

  • Transport of specific nutrients required during symbiosis

  • Cell surface remodeling during different growth phases

To functionally characterize Mnod_6500, these experimental approaches would be most informative:

• Gene knockout/knockdown studies examining effects on nodulation, nitrogen fixation, and C1 metabolism

• Localization studies during different growth conditions and symbiotic stages

• Protein-protein interaction networks specific to different physiological states

• Comparative genomics across related methylotrophs that lack nodulation ability

What are the most effective purification strategies for maintaining Mnod_6500 stability and activity?

Purification of membrane proteins like Mnod_6500 requires specialized approaches to maintain native conformation and activity. Based on membrane protein biochemistry principles, the following multi-step strategy is recommended:

1. Membrane Extraction and Solubilization:

Begin with detergent screening to identify optimal solubilization conditions. A 2D screening approach testing:

• Multiple detergent types at different concentrations

• Various buffer compositions (pH 6.0-8.0)

• Different ionic strengths

• Addition of stabilizing lipids (e.g., cholesterol, specific phospholipids)

2. Affinity Chromatography:
Leverage the N-terminal His-tag for initial purification using IMAC (Immobilized Metal Affinity Chromatography) :

• Use Ni-NTA or TALON resins

• Include low concentrations of detergent in all buffers (typically at 2-3× CMC)

• Consider gradient elution for higher purity

• Add glycerol (5-10%) to enhance stability

3. Size Exclusion Chromatography:
A critical polishing step that also provides information about protein homogeneity:

• Pre-equilibrate column with appropriate detergent buffer

• Monitor monodispersity via multi-angle light scattering (MALS)

• Collect fractions based on UV absorbance and confirmed by SDS-PAGE

4. Stability Assessment and Optimization:

• Thermal stability assays (e.g., nanoDSF, CPM assay)

• Time-course stability under various storage conditions

• Testing stabilizing additives (specific lipids, ligands, glycerol)

5. Reconstitution Options for Functional Studies:

• Proteoliposomes for transport or functional assays

• Nanodiscs for maintaining a native-like lipid environment

• Amphipols for enhanced stability in detergent-free conditions

For Mnod_6500 specifically, maintaining sample temperature at 4°C throughout purification and limiting exposure to air are recommended based on general membrane protein handling principles .

What methodologies can be employed to study the potential roles of Mnod_6500 in symbiotic relationships with Crotalaria species?

Investigating the symbiotic functions of Mnod_6500 requires an integrated approach spanning molecular, cellular, and whole-organism studies:

1. Gene Expression Analysis:

• RT-qPCR to profile mnod_6500 expression during different stages of nodulation

• RNA-Seq comparing expression in free-living versus symbiotic states

• Promoter-reporter fusions to visualize spatial-temporal expression patterns

2. Genetic Manipulation Strategies:

• Clean deletion mutants (Δmnod_6500) to assess nodulation phenotypes

• Complementation studies with wild-type and mutated versions

• Conditional expression systems to control timing of expression

• CRISPR interference for partial knockdown if complete deletion is lethal

3. Microscopy-Based Approaches:

• Immunolocalization of Mnod_6500 in nodule sections

• Fluorescent protein fusions for live-cell imaging

• Electron microscopy to examine ultrastructural localization

• Super-resolution microscopy for precise membrane distribution patterns

4. Plant-Microbe Interaction Assays:

• Nodulation efficiency assays comparing wild-type and mutant strains

• Nitrogen fixation measurement via acetylene reduction assay

• Metabolite profiling of nodules using LC-MS or GC-MS

• Competitive nodulation assays with mixed inoculations

5. Biochemical Approaches:

• Pull-down assays to identify plant proteins interacting with Mnod_6500

• Transport assays using reconstituted proteoliposomes

• Electrophysiology to assess potential channel/transporter functions

• Metabolite binding assays to identify potential substrates

6. Computational Analyses:

• Homology modeling based on related membrane proteins

• Molecular dynamics simulations in membrane environments

• Evolutionary analyses to identify conservation patterns in nodulating species

A systematic study should start with expression analysis to determine when and where Mnod_6500 is most active during symbiosis, followed by genetic manipulation to establish its requirement for successful nodulation and nitrogen fixation. Subsequently, more detailed biochemical and structural studies can elucidate its precise molecular function .

How can heterologous expression conditions be optimized to increase the yield of correctly folded Mnod_6500?

Optimizing expression of membrane proteins like Mnod_6500 requires systematic variation of multiple parameters to overcome common challenges such as toxicity, inclusion body formation, and misfolding. The following comprehensive optimization strategy is recommended:

1. Expression System Selection:

2. Vector and Fusion Design:

• Test different promoter strengths (T7, tac, ara)

• Compare N- vs C-terminal His-tags

• Evaluate fusion partners (MBP, SUMO, Mistic, GFP)

• Include cleavage sites for tag removal

3. Induction Parameter Optimization:

• Temperature (16°C, 20°C, 25°C, 30°C)

• Inducer concentration matrix

• Cell density at induction (OD600 0.4-1.0)

• Induction duration (3h, 6h, overnight)

4. Media and Additives:

• Complex vs defined media

• Supplementation with specific phospholipids

• Addition of chemical chaperones (glycerol, sucrose)

• Inclusion of ligands or stabilizing compounds

5. Systematic Screening Approach:

• Begin with small-scale expression tests (10-50 mL)

• Analyze by Western blot and in-gel fluorescence (if GFP fusion)

• Assess membrane integration vs inclusion body formation

• Scale up optimal conditions (1-10 L)

6. Quality Control Metrics:

• Monodispersity on size exclusion chromatography

• Thermal stability assays (nanoDSF, CPM assay)

• Functional assays where possible

• Circular dichroism to confirm secondary structure

For Mnod_6500 specifically, based on general membrane protein expression principles and the available information about successful E. coli expression , a promising starting point would be:

• BL21(DE3) or C41(DE3) strain

• pET vector with T7 promoter

• Induction at OD600 0.6-0.8

• 0.1-0.5 mM IPTG

• Post-induction temperature of 20°C for 16-18 hours

• TB or EnPresso medium for high cell density

This systematic approach maximizes the likelihood of identifying conditions that yield correctly folded, functional Mnod_6500 protein suitable for downstream structural and functional studies.

What are the critical considerations for designing crystallization trials for structure determination of Mnod_6500?

Crystallizing membrane proteins like Mnod_6500 presents unique challenges requiring specialized approaches. A comprehensive crystallization strategy should address:

1. Pre-Crystallization Sample Preparation:

Sample homogeneity is critical for successful crystallization. Implement these quality control measures:

• Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)

• Negative-stain electron microscopy to assess particle uniformity

• Thermal stability assays to identify optimal buffer conditions

• Dynamic light scattering to monitor aggregation propensity

2. Crystallization Method Selection:

Given the relatively small size of Mnod_6500 (107 amino acids) , both traditional vapor diffusion and LCP methods should be attempted.

3. Detergent and Lipid Optimization:

• Screen multiple detergents focusing on those successful for similar-sized membrane proteins

• Test detergent mixtures which can improve crystal contacts

• For LCP trials, test different monoacylglycerols (MAGs) and lipid additives

• Consider cholesterol or specific phospholipids based on native membrane composition

4. Systematic Screening Strategy:

• Initial broad screening using commercial sparse matrix screens

• Grid screens around promising conditions

• Additive screens to improve crystal quality

• Optimization of drop sizes, ratios, and temperatures

5. Protein Engineering Approaches:

• Construct design with variable N- and C-terminal truncations

• Surface entropy reduction to create crystal contacts

• Fusion with crystallization chaperones (e.g., T4 lysozyme, BRIL)

• Thermostabilizing mutations based on computational prediction

6. Data Collection Considerations:

• Prepare for microcrystals that may require microfocus beamlines

• Plan for serial crystallography if crystals are small

• Consider room temperature data collection to capture physiologically relevant conformations

7. Alternative Approaches if Crystallization Proves Challenging:

• Cryo-EM for structure determination without crystals

• NMR studies for dynamic regions and ligand binding

• Integrative structural biology combining low-resolution techniques

For Mnod_6500, initial screening should focus on conditions successful for other small membrane proteins from the UPF0060 family or structurally characterized prokaryotic membrane proteins of similar size .

How can computational methods enhance our understanding of Mnod_6500 structure and function?

Computational approaches offer powerful insights into membrane proteins like Mnod_6500, especially when experimental structural data is limited. An integrated computational strategy includes:

1. Sequence-Based Analysis:

• Profile-based homology detection using HHpred and HMMER

• Evolutionary coupling analysis to predict residue contacts (EVfold, GREMLIN)

• Transmembrane topology prediction (TMHMM, Phobius, TOPCONS)

• Functional site prediction using conservation patterns (ConSurf, Evolutionary Trace)

2. Structure Prediction:

• AlphaFold2 or RoseTTAFold for initial structural models

• Specialized membrane protein-specific refinement (e.g., Memoir)

• Model validation using ProQ3D or QMEANBrane for membrane proteins

• Generation of ensemble models to capture conformational flexibility

3. Molecular Dynamics Simulations:

• All-atom simulations in explicit membrane environments (CHARMM-GUI)

• Coarse-grained simulations for longer timescales (MARTINI force field)

• Enhanced sampling techniques to explore conformational landscape

• Analysis of stable water networks, lipid interactions, and dynamic properties

4. Protein-Protein Interaction Prediction:

• Membrane protein docking (e.g., HADDOCK-membrane)

• Coevolutionary approaches to identify interaction partners

• Prediction of binding sites based on surface properties

5. Functional Annotation:

• Ligand binding site prediction (SiteMap, FTMap)

• Transport pathway analysis (HOLE, CAVER)

• Electrostatic analysis for potential ion conduction paths

• Comparison with membrane protein design principles from DeGrado Lab approaches

6. Integration with Experimental Data:

• Refinement of models using low-resolution experimental constraints

• Validation of predicted structures using targeted mutagenesis

• Design of experiments based on computational hypotheses

For Mnod_6500 specifically, beginning with AlphaFold2 prediction followed by refinement in an explicit membrane would provide a foundational structural model. This can guide experimental design for functional characterization and potentially reveal structural similarities to membrane proteins of known function, despite limited sequence homology .

What controls should be included when studying the effects of Mnod_6500 on biological systems?

1. Genetic Controls:

2. Protein-Level Controls:

• Tag-only control protein to distinguish tag effects from protein function

• Denatured protein control to differentiate specific from non-specific effects

• Related but functionally distinct membrane protein as specificity control

• Varying concentrations to establish dose-response relationships

3. System-Specific Controls:

For nodulation studies:

• Non-nodulating Methylobacterium species control

• Nodulation with different Crotalaria species to assess host specificity

• Time-course controls to account for developmental variation

• Environmental controls (temperature, humidity, light conditions)

For protein interaction studies:

• Known non-interacting membrane protein as negative control

• Validated interacting pairs as positive control

• Control for detergent effects on apparent interactions

• Competition controls with unlabeled protein

4. Technical Controls:

• Multiple biological replicates (minimum n=3)

• Technical replicates to assess method variability

• Blinding procedures for phenotypic scoring

• Randomization of sample processing order

5. Validation Through Orthogonal Methods:

• Confirm key findings using independent techniques

• Vary experimental conditions to test robustness of observations

• Cross-validate in different experimental systems where feasible

For studying Mnod_6500 specifically, an ideal control set would include both loss-of-function approaches (gene deletion) and gain-of-function approaches (heterologous expression in related non-nodulating methylotrophs) to comprehensively assess its role in symbiosis and membrane biology .

How can studies of Mnod_6500 contribute to our understanding of membrane protein evolution and design?

Research on Mnod_6500 offers unique insights into membrane protein evolution and design principles, particularly at the intersection of methylotrophy and symbiotic nitrogen fixation:

1. Evolutionary Insights:

Methylobacterium nodulans represents a fascinating evolutionary case study as it combines methylotrophic metabolism with nitrogen-fixing capabilities, a combination rare in bacteria . Studying Mnod_6500 can illuminate:

• Evolutionary trajectories leading to novel membrane protein functions in specialized niches

• Molecular adaptations enabling dual lifestyle (free-living vs. symbiotic)

• Horizontal gene transfer patterns in membrane proteins across bacterial lineages

• Selection pressures shaping membrane protein sequences in plant-associated bacteria

Comparative genomic approaches can reveal:

• Conservation patterns across different Methylobacterium species with varying host ranges

• Synteny analysis to identify gene clusters suggesting functional relationships

• Positive selection signatures indicating adaptive evolution

• Convergent evolution with other plant-associated bacteria

2. Membrane Protein Design Applications:

The DeGrado Lab's principles of membrane protein design can be applied to and informed by Mnod_6500 studies :

• Structure-function relationships in small membrane proteins

• Design rules for helical packing in membrane environments

• Principles of membrane protein stability and folding

• Strategies for engineering new functions into existing membrane protein scaffolds

3. Research Methodology Development:

Mnod_6500 can serve as a model system for developing:

• Improved expression and purification protocols for small membrane proteins

• Novel assays for membrane protein function in symbiotic contexts

• Biophysical methods to study membrane protein-lipid interactions

• Computational tools for predicting membrane protein localization and topology

4. Integration with Synthetic Biology Approaches:

Knowledge derived from Mnod_6500 could inform:

• Design of synthetic membrane proteins for specific functions in bacteria

• Engineering of improved plant-microbe interfaces for agricultural applications

• Creation of biosensors based on membrane protein scaffolds

• Development of minimal membrane proteomes for synthetic cells

By combining structural biology, molecular evolution, and protein design principles, studies of Mnod_6500 can bridge fundamental membrane protein science with applications in agriculture and biotechnology .

What experimental approaches can determine if Mnod_6500 forms oligomeric structures in membranes?

Determining the oligomeric state of membrane proteins like Mnod_6500 requires multiple complementary approaches to overcome the challenges of membrane environments. A comprehensive strategy includes:

1. In vitro Biochemical Methods:

2. Structural Biology Approaches:

• X-ray crystallography revealing crystal packing arrangements

• Cryo-EM particle classification to identify different oligomeric states

• Solid-state NMR distance measurements between protomers

• Single-particle tracking to detect oligomer formation in membranes

3. Fluorescence-Based Methods:

• Förster Resonance Energy Transfer (FRET) between differently labeled protomers

• Fluorescence Correlation Spectroscopy (FCS) to measure diffusion properties

• Number and Brightness (N&B) analysis to determine oligomer size

• Total Internal Reflection Fluorescence (TIRF) microscopy for single-molecule counting

4. Functional Assays:

• Dominant negative mutant effects requiring oligomerization

• Complementation between inactive mutants (functional rescue)

• Concentration-dependent activity changes indicating cooperativity

• Electrophysiology revealing cooperative gating behavior (if applicable)

5. Computational Prediction and Validation:

• Interface prediction using evolutionary coupling analysis

• Molecular dynamics simulations of oligomer stability

• Symmetry-based modeling of potential oligomeric arrangements

• Comparison with structurally similar membrane proteins of known oligomeric state

Experimental Design Considerations:

For Mnod_6500 specifically, a stepwise approach would be most informative:

• Initial SEC-MALS analysis in different detergents to assess oligomeric tendency

• Validation using in situ approaches like FRET or crosslinking in native membranes

• Structural characterization of confirmed oligomeric assemblies

• Functional studies connecting oligomerization to biological activity

This multi-faceted approach accommodates the technical challenges of membrane protein biochemistry while providing multiple lines of evidence regarding the oligomeric state of Mnod_6500 .