Recombinant Bdellovibrio bacteriovorus Large-conductance mechanosensitive channel (mscL)
Description
Introduction to Recombinant Bdellovibrio bacteriovorus Large-conductance Mechanosensitive Channel (MscL)
The Recombinant Bdellovibrio bacteriovorus Large-conductance Mechanosensitive Channel (MscL) is a protein channel derived from the bacterium Bdellovibrio bacteriovorus, which is an obligate predator of Gram-negative bacteria. MscL channels are crucial for maintaining cellular integrity by acting as pressure-relief valves during osmotic stress. They are widely studied in various microbial species for their role in mechanosensation and potential applications in biotechnology and medicine.
Structure and Function of MscL Channels
MscL channels are homopentamers, meaning they consist of five identical subunits, each containing two transmembrane helices (TM1 and TM2) that play a key role in sensing membrane tension and triggering channel opening . When the cell membrane is stretched due to osmotic shock, MscL channels open to allow the efflux of water and ions, thereby preventing cell lysis. The open state of MscL channels exhibits a large conductance of approximately 3 nS, allowing for the passage of small proteins and metabolites .
Research Findings on MscL Channels
Research on MscL channels has provided insights into their structural dynamics and gating mechanisms. Studies using structural biology techniques have revealed significant conformational changes in the channel's domains during opening, including tilting of the transmembrane helices and rearrangement of the periplasmic loop . These findings suggest a highly coordinated mechanism for sensing mechanical forces and responding to osmotic stress.
Biotechnological and Therapeutic Potential
The large conductance and mechanosensitive properties of MscL channels make them attractive for biotechnological applications, such as the development of novel drug delivery systems and antimicrobial agents . Additionally, the use of Bdellovibrio bacteriovorus itself as a therapeutic agent against antibiotic-resistant bacteria has shown promise in treating infections .
Suppliers and Preparation
Recombinant Bdellovibrio bacteriovorus MscL is available from suppliers such as CUSABIO TECHNOLOGY LLC, which offers recombinant proteins for research purposes .
Product Specs
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Target Background
KEGG: bba:Bd0162
STRING: 264462.Bd0162
Q&A
What is Bdellovibrio bacteriovorus and why is its MscL channel of research interest?
Bdellovibrio bacteriovorus is a small Deltaproteobacterium distinguished by its unique predatory lifestyle, where it invades and grows within the periplasm of other Gram-negative bacteria . This bacterial predator has a biphasic lifecycle consisting of a free-swimming attack phase followed by an intraperiplasmic growth phase within its prey .
The large-conductance mechanosensitive channel (MscL) is a membrane protein that responds directly to membrane tension, allowing the efflux of solutes when cells experience osmotic challenges . While MscL has been extensively characterized in model organisms like Escherichia coli, its structure and function in predatory bacteria such as B. bacteriovorus represent a unique research opportunity. Studying MscL from B. bacteriovorus can provide insights into how mechanosensation operates in organisms with specialized lifestyles that involve dramatic membrane remodeling during prey invasion and intracellular growth .
How does the MscL structure in B. bacteriovorus compare to well-characterized MscL proteins from other bacteria?
Based on comparative analysis with well-characterized MscL proteins, the B. bacteriovorus MscL is expected to maintain the core structural features while potentially exhibiting adaptations related to its predatory lifestyle. Typical bacterial MscL proteins consist of approximately 136 amino acid residues (15 kDa) with two highly hydrophobic transmembrane domains .
The channel functions as a homohexamer in which the transmembrane α-helices undergo an iris-like expansion when the membrane experiences tension . While the M1 and M2 transmembrane domains are highly conserved across bacterial species, variations in the cytoplasmic and periplasmic regions may reflect adaptations to different ecological niches.
In E. coli, crosslinking studies have shown that the M1 helices rearrange significantly during channel gating, while interactions between M1 and M2 helices of adjacent subunits remain relatively unaltered . Similar studies with recombinant B. bacteriovorus MscL would be necessary to determine if its gating mechanism follows the same pattern or exhibits predator-specific modifications.
What expression systems are most suitable for producing recombinant B. bacteriovorus MscL?
For recombinant expression of membrane proteins like MscL from B. bacteriovorus, several expression systems can be considered, each with specific advantages for different research objectives:
| Expression System | Advantages | Limitations | Recommended Applications |
|---|---|---|---|
| E. coli | High yield, rapid growth, genetic tractability | Potential toxicity, inclusion body formation | Initial structural studies, mutagenesis screening |
| Cell-free systems | Avoids toxicity issues, direct incorporation into synthetic lipid environments | Lower yields, higher cost | Functional studies requiring defined lipid composition |
| Mammalian cells | Native-like post-translational modifications | Complex protocols, lower yields | Functional studies in eukaryotic membrane environment |
| B. bacteriovorus host-independent derivatives | Native cellular environment | Challenging cultivation, lower yields | Native interaction studies |
For most basic research applications, E. coli expression systems represent the optimal starting point, particularly with strains like C41(DE3) or C43(DE3) that are engineered for membrane protein expression. These systems have been successfully used for mechanosensitive channels from other bacteria and would likely accommodate the B. bacteriovorus MscL .
How can site-directed mutagenesis be optimized to identify key functional residues in B. bacteriovorus MscL?
Site-directed mutagenesis studies of B. bacteriovorus MscL should focus on evolutionary conserved residues identified through comparative genomic analysis with well-characterized MscL proteins. Based on studies with other bacterial MscL channels, several approaches would be most effective:
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Transmembrane domain mutations: Substitutions in the M1 helices, particularly at positions analogous to residues 20 and 36 in E. coli MscL, can be used to trap the channel in specific conformational states . Cysteine substitutions at these positions would allow for disulfide crosslinking studies to characterize conformational changes during gating.
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Periplasmic loop modifications: Mutations of residues in the periplasmic loop, particularly those corresponding to glutamine residues identified as critical in other bacteria, can significantly alter channel kinetics and mechanosensitivity . The following experimental design would be recommended:
| Mutation Type | Target Residues | Expected Effect | Analysis Method |
|---|---|---|---|
| Conservative substitutions | Hydrophobic M1 residues | Altered threshold tension | Patch-clamp in spheroplasts |
| Charge reversals | Lysine in M1 domain | Shifted mechanosensitivity | Liposome reconstitution assays |
| Cysteine pairs | Adjacent M1 helices | Conformational trapping | Crosslinking + functional assays |
| Deletions | C-terminal (up to 27 aa) | Minimal effect on core function | Comparative activity analysis |
For comprehensive mutagenesis studies, combining both in vitro functional assays with structural analysis methods like tryptophan fluorescence measurements would provide the most robust characterization of structure-function relationships .
How does the lipid environment affect the gating properties of recombinant B. bacteriovorus MscL?
The lipid environment significantly influences MscL gating properties, and this may be particularly relevant for B. bacteriovorus MscL given the bacterium's unique lifecycle involving transitions between free-swimming and intraperiplasmic states . To investigate these effects, researchers should consider:
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Membrane thickness effects: B. bacteriovorus transitions between environments with different membrane properties during its lifecycle. Reconstitution studies using lipids of varying acyl chain lengths can reveal how hydrophobic mismatch affects channel gating tension.
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Lipid-protein interactions: Specific regions of MscL interact directly with membrane lipids, with particular importance for the cytoplasmic end of the second transmembrane helix . Systematic characterization of these interactions should include:
| Lipid Composition | Analysis Technique | Parameters to Measure |
|---|---|---|
| Varying PE:PG:CL ratios | Patch-clamp electrophysiology | Gating threshold pressure |
| Asymmetric bilayers | Fluorescence resonance energy transfer | Conformational changes |
| Bilayers with tension gradients | Single-channel recordings | Open probability, conductance |
| Native B. bacteriovorus lipids | Comparative functional analysis | Evolutionary adaptation effects |
Recent studies suggest that regions acting as membrane anchors during transmembrane domain tilting are crucial for MscL gating . For B. bacteriovorus MscL, these regions may have evolved specific adaptations related to the predatory lifestyle and could represent novel targets for functional modification.
What role might MscL play in the predatory lifecycle of B. bacteriovorus?
The mechanosensitive channel MscL may serve specialized functions in B. bacteriovorus related to its predatory lifestyle. Given the bacterium's lifecycle, MscL could potentially contribute to:
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Osmotic protection during prey invasion: When B. bacteriovorus penetrates its prey, it experiences significant environmental changes that likely involve osmotic challenges . MscL could help maintain cellular integrity during this transition.
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Nutrient acquisition: B. bacteriovorus degrades prey content in a highly coordinated manner . MscL channels could potentially facilitate the uptake of specific degradation products.
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Bdelloplast formation: The transformation of prey into a rounded bdelloplast structure involves peptidoglycan modifications . MscL might respond to mechanical forces during this process.
Experimental approaches to investigate these potential roles would include:
| Research Question | Experimental Approach | Expected Outcomes |
|---|---|---|
| MscL expression during predation | Proteomic analysis at different lifecycle stages | Identification of stage-specific expression patterns |
| Role in prey invasion | Gene knockout/complementation studies | Altered predation efficiency |
| Response to bdelloplast formation | Time-lapse microscopy with fluorescent MscL | Visualization of channel redistribution |
| Contribution to osmotic stability | Osmotic challenge assays with MscL mutants | Differential survival under osmotic stress |
The proteomic approach applied to synchronous cultures of B. bacteriovorus has already revealed cell cycle-dependent protein expression , and similar methods could identify the temporal expression pattern of MscL during predation.
What are the key challenges in electrophysiological characterization of recombinant B. bacteriovorus MscL?
Electrophysiological characterization of recombinant B. bacteriovorus MscL presents several technical challenges that require specific methodological approaches:
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Patch-clamp recordings: The standard method for characterizing mechanosensitive channels involves patch-clamp recordings with applied suction pressure . For B. bacteriovorus MscL, researchers should consider:
| Challenge | Solution | Technical Considerations |
|---|---|---|
| Forming gigaseals with recombinant membranes | Optimize lipid composition | Include 25-30% negatively charged lipids |
| Applying calibrated pressure | Use high-precision pressure clamps | Record pressure-response curves with multiple replicates |
| Distinguishing MscL from endogenous channels | Use purified protein in liposomes | Establish protein-to-lipid ratios that favor single-channel recordings |
| Measuring large conductance | Adjust recording solutions | Use lower ionic strength to reduce current magnitudes |
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Single-channel analysis: The large conductance of MscL channels (approximately 2.5 nS) requires careful experimental design . Recordings should be made at different membrane potentials to construct current-voltage relationships, with attention to symmetrical and asymmetrical ion conditions to characterize selectivity properties.
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Correlating structure with function: Combining electrophysiology with structural modifications through mutagenesis enables mapping of the channel's functional domains. Particularly informative are experiments that couple cysteine substitutions with thiol-reactive compounds to modify channel properties in real-time during recordings.
How can advanced imaging techniques be applied to study B. bacteriovorus MscL in its native context?
Advanced imaging approaches offer powerful tools for investigating B. bacteriovorus MscL in its native cellular context during predation:
| Imaging Technique | Application | Expected Insights |
|---|---|---|
| Super-resolution microscopy | Tracking MscL distribution during predation | Spatial reorganization during lifecycle stages |
| FRET-based tension sensors | Monitoring membrane tension changes | Correlation between tension and channel activation |
| Single-molecule tracking | Following MscL molecules in living cells | Diffusion dynamics during predatory lifecycle |
| Cryo-electron tomography | Visualizing MscL in bdelloplasts | Structural context within the predator-prey interface |
For these approaches, genetic tools for creating fluorescent protein fusions with MscL would need to be optimized for B. bacteriovorus. Recent advances in synchronization methods for B. bacteriovorus cultures provide an excellent foundation for time-resolved imaging studies . By capturing images at defined timepoints (1, 2, and 3 hours after mixing with prey), researchers can create a temporal map of MscL distribution and activity throughout the predation cycle.
What computational approaches can predict the unique structural features of B. bacteriovorus MscL?
Computational methods offer valuable insights into the structural and functional properties of B. bacteriovorus MscL before experimental validation:
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Molecular dynamics simulations: Simulations of MscL embedded in lipid bilayers can predict gating mechanisms under membrane tension. For B. bacteriovorus MscL, these should incorporate:
| Simulation Parameter | Specific Considerations | Expected Outcomes |
|---|---|---|
| Membrane composition | Model both free-living and intraperiplasmic environments | Differential gating thresholds |
| Applied lateral tension | Range from 5-15 mN/m | Conformational transitions |
| Simulation timescale | Microsecond-range simulations | Complete gating events |
| Water and ion permeation | Inclusion of explicit solvent | Conductance predictions |
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Finite element modeling: This approach has been specifically applied to mechanosensitive channels to understand how membrane deformations affect channel gating . For B. bacteriovorus MscL, finite element models could predict how the unique membrane environment during prey invasion affects channel function.
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Homology modeling and protein-protein interaction predictions: These approaches can identify potential interactions between MscL and other B. bacteriovorus proteins, particularly those involved in predation. Such predictions could guide experimental designs to investigate MscL's role in the predatory lifecycle.
