Recombinant Ralstonia pickettii Large-conductance mechanosensitive channel (mscL)

What is Ralstonia pickettii MscL and what is its basic function?

Ralstonia pickettii MscL is a large-conductance mechanosensitive channel that acts as a turgor pressure release valve in bacteria. Like other MscL proteins, it undergoes large-scale conformational transitions driven directly by tension in the cytoplasmic membrane, allowing the bacterium to respond to osmotic challenges by releasing solutes when necessary . This membrane protein belongs to the family of mechanosensitive channels that are activated by mechanical stimuli, specifically membrane tension. The channel opens in response to increased membrane tension, forming a large pore that allows the passage of ions and small molecules across the membrane.

How does R. pickettii MscL compare to MscL from other bacterial species?

MscL channels are conserved across various bacterial species, including the well-studied homologues from E. coli and M. tuberculosis. Based on mechanistic studies of MscL channels, they share fundamental principles of mechanosensation, but may differ in specific gating parameters. Molecular phylogenetic analysis of R. pickettii genomic data reveals characteristic conservation patterns within the MscL family .

The tension sensitivity (defined by the tension at which half the channels are open, σ₁/₂) and the effective protein expansion during gating (ΔA) are key parameters that may vary between species. For example, E. coli MscL has been reported to have a σ₁/₂ of approximately 11.8 mN/m and a ΔA of 6.5 nm² . Comparative studies would be needed to determine these specific parameters for R. pickettii MscL.

What expression systems are effective for recombinant R. pickettii MscL production?

Recombinant R. pickettii MscL is typically expressed in E. coli expression systems. The full-length protein (amino acids 1-142) can be expressed with an N-terminal His-tag . While the provided literature doesn't detail the specific expression vectors or E. coli strains, standard bacterial expression systems used for membrane proteins would likely be suitable, such as BL21(DE3) or C43(DE3) strains with expression vectors containing T7 promoters.

The expression protocol should include:

• Transformation of the expression construct into the E. coli host

• Growth of bacterial culture to optimal density (typically OD₆₀₀ of 0.6-0.8)

• Induction of protein expression (commonly with IPTG for T7-based systems)

• Harvesting cells after appropriate expression period

• Membrane fraction isolation through cellular disruption and differential centrifugation

What purification strategies yield high-purity, functional R. pickettii MscL?

The His-tagged R. pickettii MscL can be purified using immobilized metal affinity chromatography (IMAC) . A systematic purification approach would include:

• Solubilization of membranes using appropriate detergents (typically DDM or LDAO for MscL proteins)

• IMAC purification using Ni-NTA or similar matrices

• Size exclusion chromatography for further purification and to ensure homogeneity

• Quality control by SDS-PAGE analysis (the recombinant protein should show >90% purity)

The purified protein is typically obtained as a lyophilized powder that requires appropriate reconstitution before functional studies .

What are the optimal storage conditions for purified R. pickettii MscL?

According to specifications, purified R. pickettii MscL should be stored at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles . The lyophilized protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

For reconstituted protein, it is recommended to:

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

• Add glycerol to a final concentration of 5-50% (with 50% being the default recommendation)

• Aliquot for long-term storage at -20°C/-80°C

For working aliquots, storage at 4°C for up to one week is acceptable, but repeated freezing and thawing is not recommended .

What electrophysiological approaches are most effective for studying R. pickettii MscL activity?

Electrophysiological characterization of MscL channels typically employs patch-clamp techniques in either cell-attached, inside-out, or reconstituted configurations. For R. pickettii MscL, these approaches would follow similar principles as established for other MscL homologs:

• Patch-clamp in native or reconstituted membranes: This generates robust current responses for analysis of channel conductance and gating .

• Tension application through negative pressure: Membrane tension is not directly measured but calculated from the negative pressure (suction) applied to the membrane patch using the Laplace-Young equation: σ = 2rΔP, where σ is membrane tension, r is the patch curvature, and ΔP is the applied negative pressure .

• Analysis of tension-dependent gating: The relationship between open probability (Po) and membrane tension can be fitted to determine key parameters such as the midpoint tension (σ₁/₂) and the effective protein expansion during gating (ΔA) .

• Conductance measurement: Defined as the proportionality between the voltage drop (V) across the membrane and the ionic current (I) flowing through the channel using Ohm's law .

How can molecular dynamics simulations complement experimental studies of R. pickettii MscL?

Molecular dynamics (MD) simulations provide valuable insights into MscL function that complement experimental approaches. For R. pickettii MscL research, MD simulations can:

• Predict conformational changes: Extrapolated motion techniques can predict open conformations by modifying closed structures and refining through all-atom simulations .

• Analyze pore electrostatics: MD simulations can reveal how the electrostatic environment within the channel pore influences ion conductance and selectivity .

• Simulate tension-induced gating: Application of membrane tension, steering forces, or high electric fields in simulations can reveal the mechanistic details of channel opening .

• Calculate conductance: Explicit MD and Brownian dynamics Monte Carlo simulations can be used to estimate channel conductance and compare with experimental values .

• Investigate lipid-protein interactions: Simulations can reveal how lipids interact with specific domains (such as the N-terminus) during channel expansion, leading to models like the 'dragging' model for MscL gating .

What spectroscopic methods provide structural insights into R. pickettii MscL conformational changes?

Several spectroscopic techniques have proven valuable for studying MscL conformational dynamics:

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Pulsed electron-electron double resonance (PELDOR, also known as DEER) can measure distances between spin-labeled residues to track conformational changes during gating .

  • Electron spin echo envelope modulation (ESEEM) spectroscopy can assess solvent accessibility changes of specific residues during channel activation .

  • Continuous wave EPR (cwEPR) can monitor local environmental changes and protein-lipid interactions .

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Provides information on solvent accessibility changes, albeit at lower resolution than ESEEM spectroscopy .

• Fluorescence Resonance Energy Transfer (FRET):

  • Has been used to establish helix-tilt models for MscL opening when activated by lysophosphatidylcholine (LPC) .

• Native Mass Spectrometry and Ion Mobility Mass Spectrometry:

  • Can determine the effect of detergents and lipids on channel stoichiometry and define key subconducting states in response to cysteine-specific post-translational modifications in the pore .

What key mutations affect R. pickettii MscL function and how can they be utilized in research?

While specific mutations in R. pickettii MscL are not extensively documented in the provided literature, insights from other MscL homologs suggest several potential mutations of interest:

• Gain-of-function mutations in pore-lining residues:

  • G22S mutation creates a channel with a lower activation threshold than wild-type MscL, making it approximately 30% easier to open .

  • G22E mutation creates a spontaneously active channel useful for incorporation studies .

• Mutations for chemical modulation:

  • G22C mutation allows attachment of sulfhydryl-reactive modulators to create channels responsive to various stimuli such as pH and light .

• Mutations affecting lipid sensing:

  • L89W mutation (in TbMscL) at the entrance to transmembrane pockets stabilizes an expanded and subconducting state .

These mutations could be introduced into R. pickettii MscL through standard site-directed mutagenesis techniques, providing valuable tools for investigating channel function and potential biotechnological applications.

How can R. pickettii MscL be functionally reconstituted for biophysical studies?

Functional reconstitution of R. pickettii MscL for biophysical studies typically involves:

• Liposome reconstitution:

  • Mixing purified protein with lipids (typically phosphatidylcholine and phosphatidylethanolamine mixtures)

  • Detergent removal through dialysis, Bio-Beads, or controlled dilution

  • Formation of proteoliposomes with incorporated channels

• Droplet hydrogel bilayers (DHBs):

  • A novel platform for studying mechanosensitive channels where optimization of channel incorporation can be achieved using spontaneously active mutants like MscL-G22E .

  • Buffer injection into the droplet can induce tension in the DHB sufficient to activate channels like MscL-G22S .

• Planar lipid bilayers:

  • Formation of artificial bilayers across apertures in hydrophobic supports

  • Incorporation of purified channels through fusion or direct addition

• Nanodiscs:

  • Incorporation of channels into nanometer-scale lipid bilayers stabilized by membrane scaffold proteins

  • Provides a more native-like membrane environment than detergent micelles

The reconstitution buffer should be carefully optimized for pH, ionic strength, and other parameters to maintain channel stability and function.

What methods can activate R. pickettii MscL in experimental settings?

Several approaches can be used to activate R. pickettii MscL in experimental settings:

• Mechanical activation:

  • Application of negative pressure (suction) to membrane patches in patch-clamp experiments to induce membrane tension .

  • Injection of buffer into droplets containing DHBs to stretch the inner monolayer and activate channels .

• Chemical activation:

  • Addition of amphipaths like lysophosphatidylcholine (LPC) that insert into one leaflet of the membrane, creating asymmetric bilayer tension.

  • For mutated channels with engineered cysteine residues (e.g., G22C), application of specific chemical modifiers that react with the cysteine to trigger channel opening .

• Engineered activation:

  • Light-activated variants can be created through attachment of photosensitive molecules to engineered cysteine residues .

  • pH-sensitive channels can be generated through attachment of sulfhydryl-reactive modulators to mutated residues in the pore .

The choice of activation method depends on the specific experimental goals and the technical capabilities available.

How can genomic analysis enhance our understanding of R. pickettii MscL evolution and function?

Genomic analysis provides valuable insights into the evolution and functional adaptation of R. pickettii MscL:

• Phylogenetic analysis:

  • Construction of Maximum Likelihood trees using single-nucleotide polymorphisms present in single-copy orthologous gene families can reveal evolutionary relationships .

  • Bayesian analysis of population structure can categorize R. pickettii strains and correlate genetic distances with average nucleotide identity values .

• Pan-genome analysis:

  • Identification of core-genome (35.1%) versus accessory genome (49.9%) and strain-specific genes (15.0%) provides insights into conserved and variable functions .

  • Cluster of orthologous group (COG) annotation can categorize gene families by function, revealing enrichment patterns in the core genome .

• Analysis of mobile genetic elements and horizontal gene transfer:

  • Examination of genomic islands, prophages, and HGT genes can reveal mechanisms of genetic diversity and adaptation .

  • On average, each R. pickettii genome contains approximately 17.3 genomic islands, 33.4 prophages, and 1078.3 HGT genes, with significant variations between different groups .

• Identification of virulence and resistance genes:

  • Alignment of protein sequences against databases like PHI-base and CARD can identify virulence and resistance determinants .

  • Macromolecular systems like T4SS and T6SS can be detected using tools such as MacSyFinder, TXSScan, SecReT4, and SecReT6 .

What biosafety considerations must be addressed when working with R. pickettii MscL?

Working with R. pickettii and its proteins requires careful attention to biosafety due to its potential pathogenicity:

• Risk assessment:

  • R. pickettii is an opportunistic pathogen that can cause infections, particularly in immunocompromised individuals such as neonates, cancer patients, and patients in intensive care units .

  • It has been associated with bloodstream infections, sepsis, central nervous infections, and osteomyelitis .

  • Increasing reports of drug resistance, including carbapenemase production, raise additional concerns .

• Laboratory containment measures:

  • Work should be conducted in appropriate biosafety level facilities based on risk assessment.

  • Implementation of standard microbiological practices, including proper handling, disinfection, and waste disposal.

• Prevention of contamination:

  • R. pickettii can survive in various water sources, including bottled water, purified water systems, and hospital water supplies .

  • It can form biofilms in plastic industrial water pipes and reproduce in intravenous treatment solutions .

  • Rigorous quality control of water and reagents used in experiments is essential.

• Prevention of laboratory-acquired infections:

  • Personal protective equipment appropriate for the work.

  • Procedures to minimize aerosol generation.

  • Regular monitoring of laboratory workers if extensive work with viable organisms is conducted.

• Decontamination procedures:

  • Effective disinfection protocols for work surfaces, equipment, and waste.

  • Validation of sterilization methods for materials potentially contaminated with R. pickettii.

How can R. pickettii MscL be utilized as a model for studying bacterial adaptation to environmental stresses?

R. pickettii MscL offers a valuable model for studying bacterial adaptation to environmental stresses:

• Osmoregulation mechanisms:

  • MscL acts as a turgor pressure release valve, allowing bacteria to respond to osmotic downshock by releasing solutes .

  • Studies of MscL can reveal how bacteria sense and respond to environmental osmotic fluctuations.

• Stress response integration:

  • Investigation of how mechanosensing through MscL integrates with other stress response pathways.

  • Analysis of regulatory networks controlling MscL expression under different environmental conditions.

• Adaptation to diverse environments:

  • R. pickettii is found in various environments, including water systems and hospital settings .

  • Studying MscL function across different R. pickettii strains can reveal adaptations to specific environmental niches.

• Biofilm formation and persistence:

  • R. pickettii forms biofilms in water systems and medical equipment .

  • Examination of MscL's role in biofilm formation and persistence under stress conditions.

• Host-pathogen interactions:

  • R. pickettii causes infections in immunocompromised hosts .

  • Investigation of MscL's potential role in virulence and host adaptation.

• Antibiotic resistance mechanisms:

  • R. pickettii has developed resistance to multiple antibiotics, including carbapenems .

  • Studies of MscL in the context of membrane stress responses to antibiotics could provide insights into resistance mechanisms.

Table 1: Comparative Analysis of MscL Channel Properties Across Bacterial Species

Table 2: Antibiotic Susceptibility Profile of Ralstonia pickettii