Recombinant Anabaena variabilis UPF0060 membrane protein Ava_2216 (Ava_2216)

What is the structural characterization of Ava_2216?

Ava_2216 is a UPF0060 family membrane protein from the cyanobacterium Anabaena variabilis. The full-length protein consists of 103 amino acids . Based on comparative analysis with other UPF0060 family proteins like MMAR_2961 from Mycobacterium marinum, Ava_2216 likely contains hydrophobic transmembrane domains characteristic of membrane proteins .

To experimentally characterize the structure of Ava_2216, researchers typically employ:

• Secondary structure prediction tools to identify alpha-helical and beta-sheet regions

• Hydropathy analysis to predict transmembrane domains

• Circular dichroism (CD) spectroscopy to experimentally confirm secondary structure elements

• Nuclear Magnetic Resonance (NMR) spectroscopy or X-ray crystallography for high-resolution structural determination

For membrane proteins like Ava_2216, structural characterization presents unique challenges due to their hydrophobic nature and requirement for lipid environments. Researchers often use detergent micelles or nanodiscs to stabilize the protein for structural studies.

What are the known functions of Ava_2216?

• Membrane integrity or organization

• Small molecule transport

• Signal transduction

• Protein-protein interactions at the membrane interface

To investigate the function of Ava_2216, researchers can employ:

• Gene knockout or knockdown studies in Anabaena variabilis to observe phenotypic changes

• Heterologous expression in model organisms like E. coli followed by functional assays

• Protein-protein interaction studies using pull-down assays, co-immunoprecipitation, or yeast two-hybrid screens

• Comparative genomics with other cyanobacterial species to identify conserved genomic contexts

The classification of Ava_2216 within the YnfA family provides additional research directions, as researchers can investigate whether Ava_2216 shares functional characteristics with better-characterized YnfA family proteins.

How is Ava_2216 classified within the UPF0060 protein family?

Ava_2216 belongs to the UPF0060 membrane protein family , which includes proteins from diverse bacterial species including cyanobacteria and mycobacteria. The UPF0060 designation indicates that this protein family has not been functionally characterized in detail.

Classification analysis typically includes:

• Sequence alignment with other UPF0060 family members to identify conserved residues

• Domain analysis to identify functional motifs

• Phylogenetic analysis to understand evolutionary relationships

• Structural comparison with characterized members of the family

The closely related UPF0060 membrane protein MMAR_2961 from Mycobacterium marinum shares domain features with Ava_2216 and is classified in the YnfA family . Researchers can use tools like InterPro, Pfam, and CDD to analyze domain architecture and classify Ava_2216 within protein family hierarchies.

What expression systems are optimal for producing recombinant Ava_2216?

Based on available information, E. coli has been successfully used as an expression host for recombinant Ava_2216 with a His-tag . When selecting an expression system for Ava_2216, researchers should consider:

• Expression host selection:

  • E. coli (BL21, Rosetta, C41/C43 strains optimized for membrane proteins)

  • Yeast systems (P. pastoris, S. cerevisiae) for eukaryotic post-translational modifications

  • Cell-free expression systems for toxic or difficult-to-express proteins

• Vector design considerations:

  • Promoter strength (T7, tac, araBAD)

  • Fusion tags (His, GST, MBP, SUMO) for purification and solubility

  • Signal sequences for membrane targeting

  • Codon optimization for the host organism

• Induction and expression conditions:

  • Temperature (often lowered to 16-25°C for membrane proteins)

  • Inducer concentration (IPTG, arabinose)

  • Expression duration

  • Media composition

A methodological approach to optimize expression would include:

For membrane proteins like Ava_2216, expression often requires careful optimization to balance protein production with proper membrane integration and folding.

What purification strategies yield the highest purity and activity for Ava_2216?

For His-tagged recombinant Ava_2216 , a multi-step purification strategy is recommended:

• Initial membrane isolation:

  • Cell lysis (sonication, French press, or detergent-based methods)

  • Differential centrifugation to isolate membrane fractions

  • Detergent solubilization (common detergents: DDM, LDAO, OG, Triton X-100)

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged Ava_2216

  • Careful optimization of imidazole concentrations in wash and elution buffers

  • Consideration of detergent critical micelle concentration (CMC) in all buffers

• Secondary purification steps:

  • Size exclusion chromatography (SEC) to separate monomeric protein from aggregates

  • Ion exchange chromatography (IEX) for further purification

  • Removal of fusion tags if necessary (TEV or thrombin protease cleavage)

Activity preservation considerations:

For membrane proteins like Ava_2216, maintaining the native-like lipid environment or transitioning to a suitable membrane mimetic (nanodiscs, amphipols, or liposomes) may be crucial for preserving structure and function throughout purification.

How can researchers verify the structural integrity of purified Ava_2216?

Verifying the structural integrity of purified Ava_2216 is essential to ensure that experimental results reflect native protein properties. Multiple complementary approaches should be employed:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure content

  • Differential scanning fluorimetry (DSF) to determine thermal stability

  • Dynamic light scattering (DLS) to evaluate monodispersity and aggregation state

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to determine oligomeric state

• Functional verification:

  • Binding assays for known interactors

  • Activity assays if enzymatic function is known

  • Reconstitution into liposomes to verify membrane integration

• Structural homogeneity assessment:

  • SDS-PAGE and native PAGE to assess purity and oligomeric state

  • Mass spectrometry to confirm protein identity and detect post-translational modifications

Thermal stability analysis using DSF, as described for AAV capsid proteins , can be adapted for membrane proteins like Ava_2216 to determine melting temperatures and assess the effects of different buffer conditions on protein stability.

For membrane proteins, native-like environments are crucial for structural integrity. Researchers may need to evaluate different membrane mimetics (detergent micelles, nanodiscs, or liposomes) to identify conditions that best preserve the native structure of Ava_2216.

What phylogenetic approaches are useful for studying Ava_2216 evolution?

Phylogenetic analysis of Ava_2216 and related UPF0060 family proteins can reveal evolutionary relationships and functional adaptations:

• Sequence collection and alignment:

  • Comprehensive collection of UPF0060 family sequences across diverse species

  • Multiple sequence alignment using algorithms optimized for membrane proteins

  • Manual curation to refine alignments, especially in transmembrane regions

• Phylogenetic tree construction:

  • Maximum Likelihood methods (RAxML, PhyML)

  • Bayesian inference (MrBayes, BEAST)

  • Distance-based methods (Neighbor-Joining)

  • Selection of appropriate evolutionary models (JTT, WAG, LG for proteins)

• Evolutionary analysis:

  • Calculation of dN/dS ratios to identify selection pressures

  • Ancestral sequence reconstruction

  • Molecular clock analyses to estimate divergence times

  • Identification of co-evolving residues using mutual information analysis

Statistical considerations for phylogenetic analysis :

When conducting phylogenetic analyses of membrane proteins like Ava_2216, researchers should be cautious about the impact of compositional bias due to the hydrophobic nature of transmembrane domains. Specialized evolutionary models that account for these biases should be considered.

Can functional insights be derived from other characterized UPF0060 proteins?

Although the UPF0060 family is generally uncharacterized, insights may be gleaned from partially characterized members or related protein families:

• Literature mining approaches:

  • Systematic review of published studies on any UPF0060 family members

  • Expansion to structurally similar membrane protein families

  • Analysis of high-throughput studies that may include UPF0060 proteins

• Structural homology-based inference:

  • Identification of proteins with similar fold but different sequence

  • Domain architecture comparison with functionally characterized proteins

  • Binding pocket analysis for potential substrate interactions

• Genomic context analysis:

  • Investigation of conserved gene neighborhoods across species

  • Correlation with metabolic pathways or stress responses

  • Co-expression patterns with genes of known function

• Experimental validation strategies:

  • Heterologous complementation studies

  • Substrate screening approaches

  • Phenotypic analysis of gene knockouts across species

The YnfA family classification provides a starting point for functional investigations. Researchers could examine known functions of YnfA proteins in model organisms and design experiments to test whether Ava_2216 shares these functions.

Researchers should recognize that functional predictions based on homology require experimental validation, particularly for understudied protein families like UPF0060.

What are the most effective approaches for studying Ava_2216 membrane integration?

As a membrane protein, understanding how Ava_2216 integrates into biological membranes is crucial for functional characterization:

• In vivo membrane integration studies:

  • GFP fusion approaches to visualize cellular localization

  • Protease accessibility assays to determine topology

  • Site-directed crosslinking to identify neighboring proteins

  • Alkaline extraction assays to distinguish peripheral from integral membrane association

• In vitro reconstitution approaches:

  • Reconstitution into liposomes of defined lipid composition

  • Nanodiscs for stable membrane protein complexes

  • Planar lipid bilayers for electrophysiological studies

  • Bicelles or amphipols as alternative membrane mimetics

• Biophysical characterization of membrane integration:

  • Förster Resonance Energy Transfer (FRET) to measure protein-lipid interactions

  • Neutron reflectometry to determine insertion depth

  • Solid-state NMR to analyze protein dynamics in membranes

  • Atomic Force Microscopy to visualize membrane proteins in lipid bilayers

Advanced experimental design for membrane integration studies:

When designing experiments to study Ava_2216 membrane integration, researchers should consider the native lipid environment of Anabaena variabilis membranes, which differ from model systems like E. coli. Adapting the membrane mimetic to reflect native lipid composition may be crucial for observing physiologically relevant behavior.

How can researchers investigate potential protein-protein interactions of Ava_2216?

Identifying protein-protein interactions is essential for understanding the functional context of Ava_2216:

• In vivo interaction detection methods:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Split-GFP complementation assays

  • In vivo crosslinking followed by co-immunoprecipitation

  • Proximity-dependent biotin identification (BioID)

• In vitro interaction analysis:

  • Pull-down assays using purified Ava_2216 as bait

  • Surface Plasmon Resonance (SPR) for kinetic and affinity measurements

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Microscale Thermophoresis (MST) for interaction studies in solution

• Large-scale interactome mapping:

  • Tandem Affinity Purification followed by mass spectrometry (TAP-MS)

  • Protein microarrays with purified Ava_2216

  • Label-free quantitative proteomics comparing wild-type and Ava_2216 knockout strains

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

Experimental design considerations for membrane protein interactions:

The choice of detergent or membrane mimetic is particularly critical when studying interactions of membrane proteins like Ava_2216, as inappropriate solubilization may disrupt native interactions. Validation of interactions through multiple independent methods is strongly recommended.

What statistical methods are appropriate for analyzing Ava_2216 experimental data?

• Experimental design and sample size determination:

  • Power analysis to determine required replicates

  • Randomization and blocking strategies to minimize bias

  • Consideration of biological and technical replicates

  • Factorial designs to investigate multiple variables simultaneously

• Data analysis approaches based on data type :

  • For normally distributed continuous data: parametric methods (t-tests, ANOVA)

  • For non-normally distributed data: non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis)

  • For categorical data: chi-square tests, Fisher's exact test

  • For time-to-event data: survival analysis methods

• Advanced statistical approaches for complex datasets:

  • Multivariate analysis for multiple dependent variables

  • Principal Component Analysis (PCA) for dimensionality reduction

  • Hierarchical clustering for identifying patterns

  • Machine learning approaches for predictive modeling

Key statistical methods and their applications in protein research :

For complex experiments involving multiple variables, such as optimization of expression conditions or membrane composition effects on Ava_2216 function, researchers should consider factorial experimental designs and appropriate multifactorial statistical analyses . Proper statistical analysis and reporting is crucial for reproducible research on understudied proteins like Ava_2216.

What are common difficulties in Ava_2216 expression and purification?

Membrane proteins like Ava_2216 present unique challenges in expression and purification that researchers should anticipate:

• Expression challenges:

  • Toxicity to host cells due to membrane disruption

  • Protein misfolding and aggregation

  • Inclusion body formation

  • Low expression levels

  • Improper membrane integration

• Purification obstacles:

  • Inefficient solubilization from membranes

  • Detergent-induced destabilization

  • Co-purification of lipids and other membrane components

  • Protein aggregation during concentration

  • Loss of structural integrity during purification steps

• Troubleshooting strategies:

  • Systematic optimization of expression conditions (temperature, inducer concentration, time)

  • Evaluation of multiple detergents for solubilization and purification

  • Use of fusion partners to enhance solubility and expression

  • Incorporation of stabilizing ligands during purification

  • Implementation of quality control checkpoints throughout the process

Methodological approaches to common challenges:

For His-tagged Ava_2216 , researchers should pay particular attention to the optimization of IMAC conditions, as membrane proteins often exhibit non-specific interactions with the resin due to exposed hydrophobic surfaces.

How can researchers address solubility issues with Ava_2216?

Solubility is a critical challenge for membrane proteins like Ava_2216, requiring specialized approaches:

• Fusion tag strategies:

  • N-terminal fusion partners (MBP, SUMO, Trx) to enhance solubility

  • Careful consideration of tag removal options and their impact on protein stability

  • Dual tagging approaches for enhanced purification specificity

  • Optimization of linker length between tag and protein

• Membrane mimetic selection:

  • Systematic screening of detergents (non-ionic, zwitterionic, and ionic)

  • Mixed micelle approaches combining primary and secondary detergents

  • Lipid-detergent mixtures to better mimic native environments

  • Alternative membrane mimetics (nanodiscs, amphipols, SMALPs)

• Buffer optimization:

  • Systematic variation of pH, ionic strength, and buffer components

  • Addition of stabilizing agents (glycerol, specific lipids, ligands)

  • Evaluation of divalent cation effects (Mg²⁺, Ca²⁺)

  • Temperature effects on solubility and stability

Systematic approach to detergent screening:

For recombinant Ava_2216 , researchers should consider the potential impact of the His-tag position on protein solubility and function. Comparing N-terminal versus C-terminal tagging, or evaluating different tag positions, may identify constructs with improved solubility properties.

What quality control measures should be implemented for Ava_2216 research?

Robust quality control is essential for ensuring reliable and reproducible results in Ava_2216 research:

• Protein identity and integrity verification:

  • Mass spectrometry confirmation of protein identity

  • N-terminal sequencing to verify the correct start site

  • SDS-PAGE and Western blotting to assess purity and integrity

  • Size exclusion chromatography to evaluate aggregation state

• Structural integrity assessment:

  • Circular dichroism to monitor secondary structure

  • Differential scanning fluorimetry to assess thermal stability

  • Dynamic light scattering to evaluate monodispersity

  • Tryptophan fluorescence to monitor tertiary structure

• Functional validation:

  • Development of activity assays specific to predicted function

  • Ligand binding studies if binding partners are known

  • Reconstitution into proteoliposomes for functional studies

  • In vivo complementation assays

• Experimental reproducibility considerations:

  • Detailed documentation of all experimental conditions

  • Establishment of positive and negative controls

  • Implementation of appropriate statistical analysis

  • Validation of key findings using multiple approaches

Quality control decision tree:

For membrane proteins like Ava_2216, additional quality control measures related to the lipid environment or detergent micelle properties may be necessary. Analytical ultracentrifugation or SEC-MALS can provide information about the protein-detergent complex size and composition, ensuring consistent preparation across experiments.