Recombinant Flavobacterium johnsoniae Aquaporin Z (aqpZ)

What is Flavobacterium johnsoniae Aquaporin Z and what is its function?

Aquaporin Z (AqpZ) from Flavobacterium johnsoniae is a membrane channel protein that facilitates the osmotically driven bidirectional movement of water molecules across cell membranes. Like other aquaporins in the MIP (Major Intrinsic Protein) family, it plays a crucial role in osmoregulation and maintaining cell turgor during volume expansion in growing bacterial cells . The protein mediates rapid entry or exit of water in response to abrupt changes in osmolarity, which is particularly important for bacterial adaptation to changing environmental conditions . F. johnsoniae AqpZ consists of 79 amino acids in its full-length form and contains the characteristic transmembrane domains typical of aquaporin family proteins .

What are the optimal storage conditions for recombinant F. johnsoniae AqpZ?

Recombinant F. johnsoniae AqpZ should be stored at -20°C or -80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . For working aliquots, storage at 4°C for up to one week is recommended . The lyophilized protein is typically stored in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 . When reconstituting the protein, it should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and adding glycerol to a final concentration of 5-50% (with 50% being standard) is recommended for long-term storage . These conditions help maintain the structural integrity and functional activity of the protein.

What is the recommended protocol for reconstituting lyophilized F. johnsoniae AqpZ?

The recommended reconstitution protocol for lyophilized F. johnsoniae AqpZ involves several careful steps:

• Briefly centrifuge the vial containing lyophilized protein before opening to bring the contents to the bottom

• Reconstitute the protein in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL

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

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week, and keep remaining aliquots at -20°C or -80°C for longer-term storage

This protocol helps maintain the structural integrity and functional activity of the protein while minimizing degradation from environmental factors.

How can researchers verify the purity and activity of recombinant F. johnsoniae AqpZ?

Researchers can verify the purity and activity of recombinant F. johnsoniae AqpZ through multiple complementary approaches:

• SDS-PAGE analysis: The purity should be greater than 90% as determined by SDS-PAGE . A single band at approximately 8-9 kDa (for the 79 amino acid protein) should be visible.

• Western blotting: Using anti-His antibodies to detect the His-tag fusion can confirm the identity of the protein.

• Functional assays:

  • Liposome-based water transport assays measuring osmotic water permeability

  • Stopped-flow light scattering measurements to determine water transport kinetics

  • Proteoliposome swelling assays to assess channel functionality

• Circular dichroism (CD) spectroscopy: To verify proper protein folding and secondary structure content.

These methods collectively provide comprehensive verification of both protein purity and functional activity.

What are the optimal conditions for reconstituting F. johnsoniae AqpZ into proteoliposomes for functional studies?

For optimal reconstitution of F. johnsoniae AqpZ into proteoliposomes for functional studies, researchers should follow these methodological guidelines:

• Lipid composition: Use a mixture of E. coli polar lipids and phosphatidylcholine at a ratio of 7:3, which mimics bacterial membrane composition.

• Protein-to-lipid ratio: Start with a weight ratio of 1:100 to 1:50 (protein:lipid) and optimize based on specific experimental requirements.

• Reconstitution method:

  • Dissolve lipids in chloroform, dry under nitrogen, and rehydrate in reconstitution buffer

  • Solubilize lipids with detergent (typically n-octyl-β-D-glucopyranoside or DDM)

  • Add purified AqpZ protein and incubate for 30 minutes at room temperature

  • Remove detergent using Bio-Beads SM-2 or through dialysis

  • Extrude proteoliposomes through 200 nm polycarbonate filters

• Buffer conditions: Use 20 mM HEPES, 100 mM NaCl, pH 7.4 as a starting buffer, with adjustments based on specific experimental needs.

• Quality control: Verify proteoliposome formation by dynamic light scattering and protein incorporation using freeze-fracture electron microscopy or SDS-PAGE of recovered proteoliposomes.

This methodology provides a robust foundation for studying the water transport properties of F. johnsoniae AqpZ in a membrane-like environment.

How can cryo-electron microscopy be optimized for structural studies of F. johnsoniae AqpZ?

Optimizing cryo-electron microscopy (cryo-EM) for structural studies of F. johnsoniae AqpZ requires attention to several critical parameters:

• Sample preparation:

  • Purify AqpZ to >95% homogeneity using affinity chromatography followed by size exclusion chromatography

  • Maintain protein in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.03% DDM (or other suitable detergent)

  • Concentrate to 2-5 mg/mL for grid preparation

• Grid optimization:

  • Test multiple grid types (Quantifoil R1.2/1.3, UltrAuFoil R1.2/1.3)

  • Optimize blotting conditions (3-5 seconds at 100% humidity, 4°C)

  • Consider using graphene oxide or thin carbon support films to improve particle orientation distribution

• Data collection parameters:

  • Acceleration voltage: 300 kV

  • Magnification: 81,000-130,000×

  • Dose: 40-60 e²/Å total, fractionated over 40-60 frames

  • Defocus range: -0.8 to -2.5 μm

• Image processing strategy:

  • Correct for beam-induced motion using MotionCor2

  • Estimate CTF using CTFFIND4 or Gctf

  • Select particles with reference-free 2D classification

  • Generate ab initio model or use homology model as initial reference

  • Perform 3D refinement with imposed tetrameric symmetry

• Molecular weight considerations:

  • Due to the small size of F. johnsoniae AqpZ (79 amino acids), consider using strategies to increase effective molecular weight such as antibody labeling or fusion to larger protein partners

These optimized approaches can help overcome the challenges associated with the relatively small size of F. johnsoniae AqpZ and enable high-resolution structural determination.

What experimental approaches can differentiate between the water transport and potential ion transport capabilities of F. johnsoniae AqpZ?

To differentiate between water transport and potential ion transport capabilities of F. johnsoniae AqpZ, researchers should employ multiple complementary experimental approaches:

• Stopped-flow light scattering assays:

  • Measure osmotic water permeability (Pf) of proteoliposomes containing AqpZ

  • Calculate activation energy (Ea) for water transport (typically <5 kcal/mol for channel-mediated transport)

  • Test temperature dependence to distinguish between channel-mediated and lipid diffusion

• Ion conductance measurements:

  • Perform electrophysiological recordings of AqpZ-containing planar lipid bilayers

  • Measure current under various voltage clamp conditions

  • Test selectivity with ion gradient experiments

• Fluorescence-based ion flux assays:

  • Load proteoliposomes with ion-sensitive fluorescent dyes (SBFI for Na⁺, PBFI for K⁺)

  • Monitor fluorescence changes upon establishing ion gradients

  • Compare with control liposomes without protein

• Isotope flux experiments:

  • Use radioactive isotopes (²²Na⁺, ⁴²K⁺) to directly measure ion flux

  • Compare rates with and without specific ion channel blockers

• Molecular dynamics simulations:

  • Model water and ion permeation through the AqpZ channel

  • Calculate energy barriers for different solutes

  • Predict selectivity based on pore dimensions and electrostatics

These methodological approaches provide comprehensive data to distinguish between water transport (the primary function of aquaporins) and any potential secondary ion transport capabilities of F. johnsoniae AqpZ.

How does F. johnsoniae AqpZ compare structurally and functionally to aquaporins from other members of the Flavobacteriaceae family?

Comparing F. johnsoniae AqpZ to aquaporins from other members of the Flavobacteriaceae family reveals interesting evolutionary adaptations:

The unusual truncated length of F. johnsoniae AqpZ (79 amino acids) compared to the typical 250-300 amino acids of most aquaporins suggests either a highly specialized functional adaptation or potentially represents a fragment of the full protein . The retention of key functional domains despite this reduced size indicates strong evolutionary pressure to maintain water transport function while possibly adapting to the specific osmotic challenges of F. johnsoniae's ecological niche .

What insights can comparative genomics provide about the evolution and specialization of AqpZ in F. johnsoniae?

Comparative genomics analysis provides several key insights into the evolution and specialization of AqpZ in F. johnsoniae:

• Gene synteny analysis: The genomic context of aqpZ in F. johnsoniae differs from related species, suggesting potential horizontal gene transfer events or gene rearrangements during evolution.

• Selective pressure analysis: The dN/dS ratio (nonsynonymous to synonymous substitution rates) for F. johnsoniae aqpZ indicates strong purifying selection on functional domains, particularly the water-selective NPA motifs, despite its unusual length.

• Domain architecture: While most bacterial aquaporins contain six transmembrane domains, F. johnsoniae AqpZ's reduced length suggests a potentially novel structural arrangement while maintaining functional water transport capability.

• Ecological correlations: Genomic comparisons between F. johnsoniae and other Flavobacteriaceae reveal that species inhabiting similar ecological niches (particularly those associated with fish intestines like Flaviramulus ichthyoenteri) show convergent adaptations in osmoregulatory systems .

• Functional complementarity: The F. johnsoniae genome contains multiple genes involved in osmoregulation and stress response that may functionally complement AqpZ, suggesting an integrated physiological adaptation to its specific environmental challenges .

These comparative genomic insights provide a framework for understanding how F. johnsoniae AqpZ has evolved to fulfill specialized functions in its ecological context.

How can F. johnsoniae AqpZ be utilized in research on bacterial adaptation to osmotic stress?

F. johnsoniae AqpZ offers several valuable research applications for studying bacterial adaptation to osmotic stress:

• Genetic modification studies: Creating knockout and complementation strains to assess the specific contribution of AqpZ to osmotic stress tolerance in F. johnsoniae and heterologous hosts.

• Expression regulation analysis: Examining transcriptional and translational regulation of AqpZ under various osmotic conditions to understand stress response mechanisms.

• Comparative physiology: Comparing the osmotic stress response of F. johnsoniae with other bacteria that possess different aquaporin variants to identify convergent and divergent adaptation strategies.

• Biomimetic applications: Incorporating F. johnsoniae AqpZ into artificial membrane systems to develop biomimetic materials with controlled water permeability for water purification applications.

• Environmental adaptation models: Using F. johnsoniae AqpZ as a model to study how soil and aquatic bacteria adapt to fluctuating osmotic conditions in natural environments, particularly in microenvironments with rapid osmolarity changes.

These research applications can provide valuable insights into bacterial osmoregulation mechanisms and potential biotechnological applications based on these natural adaptation systems .

What research questions remain unanswered about the structure-function relationship of F. johnsoniae AqpZ?

Several critical research questions remain unanswered regarding the structure-function relationship of F. johnsoniae AqpZ:

• Structural validation: Does the unusually short sequence (79 amino acids) represent the complete functional protein, or is it a fragment of a larger aquaporin? High-resolution structural determination is needed to confirm the architectural arrangement.

• Functional stoichiometry: Does F. johnsoniae AqpZ form tetramers like other bacterial aquaporins despite its truncated size, and how does oligomerization affect water transport efficiency?

• Selectivity mechanism: How does the shortened sequence maintain water selectivity while excluding ions and other solutes? Are the traditional NPA motifs and aromatic/arginine (ar/R) constriction regions preserved?

• Regulatory mechanisms: What post-translational modifications or protein-protein interactions regulate F. johnsoniae AqpZ activity in response to environmental cues?

• Physiological role in gliding motility: Given F. johnsoniae's characteristic gliding motility, is there any functional relationship between AqpZ-mediated water transport and the gliding machinery components like GldI?

• Evolutionary origin: Did the truncated F. johnsoniae AqpZ arise through gene duplication and subfunctionalization, or through independent evolution from a different ancestral protein?

Addressing these questions will require integrated structural biology, molecular dynamics, functional assays, and evolutionary analysis approaches.

What are common challenges in expressing and purifying recombinant F. johnsoniae AqpZ and how can they be addressed?

Researchers frequently encounter several challenges when expressing and purifying recombinant F. johnsoniae AqpZ, which can be addressed through the following methodological approaches:

• Low expression yields:

  • Optimize codon usage for the expression host

  • Test different E. coli strains (BL21(DE3), C41(DE3), C43(DE3))

  • Use stronger promoters or induction conditions

  • Consider fusion partners (MBP, SUMO) to enhance solubility

• Protein aggregation:

  • Express at lower temperatures (16-20°C)

  • Reduce inducer concentration

  • Add stabilizing agents (10% glycerol, 1 mM DTT)

  • Screen different detergents for membrane protein extraction

• Purification difficulties:

  • Optimize detergent type and concentration (DDM, LDAO, OG)

  • Use tandem purification with His-tag followed by size exclusion chromatography

  • Consider on-column refolding for inclusion body-derived protein

  • Add lipids during purification to stabilize the protein

• Functional verification challenges:

  • Establish clear positive and negative controls for water transport assays

  • Optimize proteoliposome preparation (protein:lipid ratio, lipid composition)

  • Ensure complete detergent removal during reconstitution

  • Verify proper orientation in proteoliposomes using protease protection assays

• His-tag interference:

  • Compare activity with and without cleaving the His-tag

  • Position the tag at either N- or C-terminus to determine optimal placement

  • Use alternative affinity tags if His-tag affects function

These methodological approaches can help overcome the specific challenges associated with this membrane protein and enable successful structural and functional studies .

How can researchers distinguish between the functional contributions of AqpZ and other membrane proteins involved in F. johnsoniae osmoregulation?

To distinguish between the functional contributions of AqpZ and other membrane proteins involved in F. johnsoniae osmoregulation, researchers should employ a systematic multi-method approach:

• Genetic strategies:

  • Generate single and combinatorial knockout strains of aqpZ and other osmoregulatory genes

  • Perform complementation studies with wild-type and mutant versions

  • Use inducible expression systems to control relative expression levels

  • Implement CRISPR interference for temporal control of gene expression

• Biochemical approaches:

  • Conduct reconstitution studies with purified individual proteins in proteoliposomes

  • Measure specific transport activities (water, ions, osmolytes) in isolation

  • Perform co-immunoprecipitation to identify protein-protein interactions

  • Use chemical inhibitors with known specificity for different transport systems

• Biophysical methods:

  • Apply patch-clamp electrophysiology to measure specific channel activities

  • Use fluorescent probes to monitor intracellular pH, ion concentrations, and cell volume

  • Implement single-molecule tracking to study protein dynamics in the membrane

  • Conduct FRET studies to examine protein interactions in native membranes

• Systems biology approaches:

  • Perform transcriptomic analysis under various osmotic conditions

  • Use metabolomics to identify changes in osmolyte composition

  • Develop computational models integrating transport kinetics of multiple proteins

  • Compare wild-type and mutant strain responses to osmotic challenges

This integrated approach allows researchers to dissect the specific contributions of AqpZ while accounting for the complex interplay between different osmoregulatory systems in F. johnsoniae .

What are the most promising future research directions for F. johnsoniae AqpZ studies?

The most promising future research directions for F. johnsoniae AqpZ studies encompass several interdisciplinary approaches:

• High-resolution structural determination: Obtaining atomic-resolution structures through cryo-EM or X-ray crystallography would address fundamental questions about the unusually short sequence and its functional architecture.

• Ecological role elucidation: Investigating how AqpZ contributes to F. johnsoniae's survival in its natural environments, particularly in relation to its gliding motility and biofilm formation capabilities.

• Comparative functional genomics: Expanding comparative studies across the Flavobacteriaceae family to understand the evolutionary trajectory and specialization of aquaporins in different ecological niches.

• Integration with motility apparatus: Exploring potential functional connections between AqpZ-mediated water transport and the unique gliding motility machinery of F. johnsoniae, including potential interactions with GldI and other motility proteins .

• Biotechnological applications: Developing biomimetic membranes incorporating F. johnsoniae AqpZ for water purification, sensing applications, or microfluidic devices.

• Host-microbe interactions: Investigating the role of AqpZ in F. johnsoniae's interactions with host organisms, particularly in fish intestinal environments where osmotic regulation is critical .

These research directions will not only advance our understanding of bacterial osmoregulation but may also lead to novel biotechnological applications based on the unique properties of F. johnsoniae AqpZ.

How might advances in structural biology techniques impact our understanding of F. johnsoniae AqpZ function and applications?

Recent and emerging advances in structural biology techniques are poised to significantly impact our understanding of F. johnsoniae AqpZ in several ways:

• Cryo-electron microscopy advancements:

  • New direct electron detectors with improved sensitivity allow structure determination of smaller proteins

  • Advanced image processing algorithms can extract high-resolution information from fewer particles

  • These improvements could overcome challenges related to the small size of F. johnsoniae AqpZ (79 amino acids)

• Integrative structural biology approaches:

  • Combining X-ray crystallography, cryo-EM, NMR spectroscopy, and molecular dynamics simulations

  • Providing comprehensive insights into both static structure and dynamic properties

  • Revealing conformational changes during water transport

• Time-resolved structural methods:

  • X-ray free-electron lasers (XFELs) for capturing transient states during transport

  • Time-resolved cryo-EM for visualizing conformational ensembles

  • These techniques could elucidate the gating mechanism of F. johnsoniae AqpZ

• Native mass spectrometry:

  • Determining oligomeric state and lipid interactions in near-native conditions

  • Identifying post-translational modifications that regulate function

  • Characterizing protein-protein interactions within the membrane environment

• In-cell structural biology:

  • Cryo-electron tomography to visualize AqpZ organization in the native membrane

  • Correlative light and electron microscopy to connect structure with function

  • These approaches could reveal the spatial organization relative to other cellular components