Recombinant Mycobacterium gilvum UPF0060 membrane protein Mflv_3127 (Mflv_3127)

What is Mflv_3127 and what are its basic structural features?

Mflv_3127 is a membrane protein belonging to the UPF0060 family, originating from Mycobacterium gilvum (strain PYR-GCK), also known as Mycobacterium flavescens (strain ATCC 700033/PYR-GCK). It is a full-length protein consisting of 108 amino acids with a multi-pass membrane protein topology, meaning it traverses the cell membrane multiple times . The protein has a complete amino acid sequence of "MVVKSALLFVLAAVLEIGGAWLVWQGFREHRGWLWVGAGVLALGAYGFVAAFQPDANFGRVLAAYGGVFVAGSLIWGMVADGFRPDRWDITGAAVCLLGVVLIMYAPR," which contains several hydrophobic regions characteristic of transmembrane domains . This sequence analysis suggests the protein likely contains multiple membrane-spanning helices that anchor it within the bacterial cell membrane, with both hydrophobic transmembrane segments and more hydrophilic regions that may face the cytoplasm or extracellular environment.

What is the subcellular localization of Mflv_3127?

Mflv_3127 is localized in the cell membrane as a multi-pass membrane protein . This localization is critical to understanding its potential function, as membrane proteins typically mediate essential cellular processes including transport, signal transduction, enzymatic catalysis, and structural support . The multi-pass nature of Mflv_3127 suggests it could be involved in facilitating the movement of molecules across the membrane, cell signaling, or membrane structural integrity. The protein's membrane localization also has important implications for experimental design, as it requires specialized approaches for expression, purification, and functional characterization compared to soluble proteins.

What databases contain information about Mflv_3127?

Researchers can access information about Mflv_3127 through several database resources. The protein is cataloged in UniProt with ID A4TB05 . Additionally, it can be found in KEGG under the identifier mgi:Mflv_3127 and in STRING under the identifier 350054.Mflv_3127 . These databases provide different types of information that can be valuable for researchers: UniProt offers detailed protein sequence and functional annotation data, KEGG places the protein in metabolic and signaling pathway contexts, and STRING provides protein-protein interaction network information. Cross-referencing these databases can provide a more comprehensive understanding of Mflv_3127's biological context and potential functions within Mycobacterium gilvum.

What expression systems are most suitable for recombinant Mflv_3127 production?

The E. coli expression system has been successfully employed for the recombinant production of Mflv_3127 . This bacterial expression platform offers advantages of rapid growth, high protein yield, and well-established protocols. For optimal expression of Mflv_3127, researchers should consider using an E. coli strain optimized for membrane protein expression, such as C41(DE3), C43(DE3), or Lemo21(DE3), which are engineered to mitigate the toxicity often associated with membrane protein overexpression. Expression conditions should be carefully optimized by testing different induction temperatures (typically 16-30°C), inducer concentrations, and induction times to balance protein yield with proper folding. The addition of an N-terminal His-tag, as implemented in commercially available constructs, facilitates purification while maintaining protein functionality .

How should I design valid experiments to study Mflv_3127 function?

When designing experiments to investigate Mflv_3127 function, follow these methodological steps:

• Begin by clearly defining your variables: independent variable (e.g., expression level of Mflv_3127), dependent variable (e.g., membrane permeability, cell growth), and control for extraneous variables .

• Formulate a specific, testable hypothesis based on UPF0060 family characteristics or predicted membrane protein functions.

• Include appropriate controls in your experimental design:

  • Negative control: Cells without Mflv_3127 expression

  • Positive control: Cells expressing a well-characterized membrane protein

  • Vector-only control: Cells expressing the empty vector without Mflv_3127

• Consider both gain-of-function approaches (overexpression of Mflv_3127) and loss-of-function approaches (gene knockout or knockdown) to comprehensively assess protein function .

• Design your experimental treatments to systematically manipulate expression levels or conditions that might affect Mflv_3127 function.

• Employ both correlational studies (observing associations between Mflv_3127 expression and cellular phenotypes) and experimental studies (introducing changes to Mflv_3127 and monitoring effects) to distinguish correlation from causation .

A well-designed experimental approach will help distinguish between mere correlation and actual causation in your findings, providing stronger evidence for Mflv_3127's functional role.

What protein purification strategies are effective for Mflv_3127?

Purification of Mflv_3127 requires specialized approaches due to its membrane protein nature. The commercially available recombinant Mflv_3127 is produced with an N-terminal 10xHis-tag , enabling affinity chromatography as the primary purification method. A recommended purification protocol includes:

• Membrane fraction isolation: After cell lysis, separate the membrane fraction by ultracentrifugation.

• Solubilization: Extract Mflv_3127 from membranes using appropriate detergents such as n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucopyranoside (OG), or digitonin, which maintain membrane protein integrity.

• Immobilized metal affinity chromatography (IMAC): Utilize the His-tag for purification using Ni-NTA or Co-NTA resin.

• Size exclusion chromatography: As a polishing step to remove aggregates and achieve higher purity.

Throughout purification, maintain the protein in a buffer containing 6% trehalose and at pH 8.0 to preserve stability . The final purified protein can be stored either as a liquid in this stabilizing buffer or lyophilized for longer-term storage, with reconstitution recommendations to a concentration of 0.1-1.0 mg/mL in deionized sterile water, plus 5-50% glycerol for long-term storage at -20°C/-80°C .

What techniques are most appropriate for structural characterization of Mflv_3127?

Structural characterization of membrane proteins like Mflv_3127 presents unique challenges due to their hydrophobic nature and requirement for lipid environments. Several complementary approaches are recommended:

• X-ray crystallography: While challenging, it can provide high-resolution structures if well-diffracting crystals can be obtained. Lipidic cubic phase (LCP) crystallization may be particularly suitable for membrane proteins like Mflv_3127.

• Cryo-electron microscopy (cryo-EM): Increasingly powerful for membrane proteins, allowing visualization without crystallization. Particularly useful if Mflv_3127 forms higher-order complexes.

• Nuclear magnetic resonance (NMR) spectroscopy: Can provide structural information in solution and about protein dynamics, though challenging for larger membrane proteins.

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS): Valuable for mapping exposed regions and conformational changes without requiring a complete structure.

• Computational modeling: Homology modeling based on structurally characterized UPF0060 family members combined with molecular dynamics simulations can predict structural features .

Each of these methods contributes different insights, and a multi-technique approach is recommended for comprehensive structural characterization of Mflv_3127.

How does the His-tag influence the structure and function of recombinant Mflv_3127?

The N-terminal 10xHis-tag used in recombinant Mflv_3127 constructs can influence both protein structure and function in several ways that researchers must consider:

The His-tag provides crucial benefits for purification, but researchers should interpret functional data with awareness of its potential effects, particularly for subtle aspects of protein function or protein-protein interactions.

What is the predicted function of Mflv_3127 based on its protein family?

• Transport function: As a multi-pass membrane protein with several predicted transmembrane domains, Mflv_3127 may facilitate the movement of ions, small molecules, or nutrients across the mycobacterial cell membrane .

• Signaling role: The protein could participate in signal transduction pathways, potentially responding to environmental changes and transmitting signals to the cell interior.

• Structural role: It might contribute to membrane integrity or organization within Mycobacterium gilvum.

Membrane proteins broadly "mediate processes that are fundamental for the flourishing of biological cells," including transport, cell communication, and enzymatic catalysis . The conserved nature of the UPF0060 family across multiple bacterial species suggests an important, though not yet fully characterized, role in bacterial physiology. Further experimental characterization is needed to definitively establish the molecular function of Mflv_3127.

What functional assays are appropriate for characterizing Mflv_3127 activity?

To investigate the functional activity of Mflv_3127, researchers should employ multiple complementary assays tailored to membrane protein characterization:

• Transport assays: If Mflv_3127 functions as a transporter, researchers can use:

  • Liposome-reconstituted protein assays with fluorescent or radioactive substrates

  • Whole-cell uptake/efflux studies comparing wild-type and Mflv_3127-overexpressing cells

  • Electrophysiological techniques such as patch-clamp if ion transport is suspected

• Binding assays:

  • Surface plasmon resonance (SPR) to detect interactions with potential ligands

  • Isothermal titration calorimetry (ITC) for thermodynamic binding parameters

  • Microscale thermophoresis (MST) for detecting subtle binding events

• Structural changes upon substrate binding:

  • Circular dichroism spectroscopy to monitor secondary structure changes

  • Fluorescence-based assays if tryptophan residues are strategically located

  • HDX-MS to identify regions with altered solvent accessibility upon binding

• In vivo functional assays:

  • Growth phenotype analysis under various conditions in knockout/overexpression strains

  • Metabolomic profiling to identify accumulated or depleted metabolites

  • Membrane integrity assays to assess structural contributions

These methodological approaches should be designed with appropriate controls as described in section 2.2, ensuring that any observed effects can be attributed specifically to Mflv_3127 function.

How does Mflv_3127 compare with UPF0060 family proteins from other mycobacterial species?

Comparative analysis of Mflv_3127 with UPF0060 family proteins from other mycobacterial species provides insights into evolutionary conservation and potential functional significance. Although the search results don't provide direct sequence comparisons, a structured analytical approach would include:

• Sequence similarity analysis:

  Species	Protein ID	Sequence Identity (%)	Sequence Similarity (%)	Conservation in Transmembrane Regions
  M. gilvum	Mflv_3127	100 (reference)	100 (reference)	Reference
  M. tuberculosis	Hypothetical	To be determined	To be determined	To be determined
  M. smegmatis	Hypothetical	To be determined	To be determined	To be determined
  M. leprae	Hypothetical	To be determined	To be determined	To be determined

• Phylogenetic analysis: Construction of a phylogenetic tree of UPF0060 family proteins across mycobacterial species to understand evolutionary relationships.

• Structural motif conservation: Identification of conserved motifs particularly in transmembrane regions, which often indicate functional importance.

• Genomic context analysis: Examination of neighboring genes which can provide clues about functional associations through operonic arrangements or consistent proximity.

Higher conservation typically suggests functional importance, while divergence may indicate species-specific adaptations. Particular attention should be paid to conservation patterns in pathogenic versus non-pathogenic mycobacteria, which might suggest relevance to virulence or survival mechanisms.

What insights can be gained by comparing membrane protein research approaches for Mflv_3127 with other bacterial membrane proteins?

Comparing research approaches for Mflv_3127 with strategies used for other bacterial membrane proteins offers valuable methodological insights:

• Expression system optimization: While E. coli is commonly used for Mflv_3127 expression , other bacterial membrane proteins sometimes require alternative hosts like Pichia pastoris, mammalian cells, or cell-free systems for proper folding and function. Researchers should evaluate whether the E. coli system provides adequate expression and proper folding for Mflv_3127.

• Structural determination approaches: Recent advances in cryo-EM have revolutionized membrane protein structural biology by eliminating the need for well-diffracting crystals . This technique might be particularly valuable for Mflv_3127 if crystallization proves challenging.

• Functional characterization strategies: The experimental approaches used for well-characterized transporters, channels, or receptors can be adapted for Mflv_3127. Substrate identification often begins with structural predictions and bioinformatic analysis of conserved binding sites.

• Lipid environment considerations: Membrane proteins function in complex lipid environments that influence their activity . Researchers should consider reconstituting Mflv_3127 in native-like lipid environments rather than detergent micelles for functional studies, following approaches proven successful with other bacterial membrane proteins.

• Integration of computational and experimental methods: Successful membrane protein research increasingly combines molecular dynamics simulations with experimental validation, an approach that could elucidate Mflv_3127 function.

By adapting established methodologies from well-characterized bacterial membrane proteins, researchers can accelerate functional understanding of Mflv_3127.

What are common challenges in working with recombinant Mflv_3127 and how can they be addressed?

Researchers working with recombinant Mflv_3127 may encounter several challenges common to membrane protein research, each requiring specific troubleshooting approaches:

• Low expression yields:

  • Optimize induction conditions (temperature, inducer concentration, time)

  • Test specialized E. coli strains designed for membrane protein expression

  • Consider using stronger or weaker promoters depending on toxicity

• Protein misfolding:

  • Lower induction temperature (16-20°C) to slow expression and promote proper folding

  • Add folding enhancers like glycerol (5-10%) to the culture medium

  • Test different detergents during extraction and purification

• Aggregation during purification:

  • Ensure complete solubilization with appropriate detergent concentrations

  • Include stabilizing agents like 6% trehalose in purification buffers

  • Maintain pH at 8.0 which has been determined optimal for stability

• Loss of activity:

  • Minimize freeze-thaw cycles as recommended in the product documentation

  • Store working aliquots at 4°C for up to one week to maintain activity

  • Consider reconstitution into liposomes or nanodiscs that better mimic native membrane environment

• Poor reproducibility:

  • Standardize all protocols from expression to storage

  • Document detailed conditions including lot numbers of reagents

  • Prepare larger batches of protein when possible to minimize batch-to-batch variation

Each of these methodological approaches addresses specific challenges inherent to membrane protein research and should be systematically tested to optimize work with Mflv_3127.

What are optimal storage and handling conditions for maintaining Mflv_3127 stability?

Maintaining stability of purified recombinant Mflv_3127 requires careful attention to storage and handling conditions. Based on manufacturer recommendations and membrane protein research practices, the following protocol is advised:

• Short-term storage (up to one week):

  • Store at 4°C in Tris/PBS-based buffer at pH 8.0 containing 6% trehalose

  • Avoid repeated temperature changes

• Long-term storage options:

  • Lyophilized form: Provides stability for up to 12 months at -20°C/-80°C

  • Liquid form in stabilizing buffer: Stable for approximately 6 months at -20°C/-80°C

  • For liquid storage, add glycerol to a final concentration of 5-50% (with 50% being optimal) before freezing

• Aliquoting recommendations:

  • Divide purified protein into single-use aliquots to avoid repeated freeze-thaw cycles

  • Ensure aliquot volumes are practical for intended experiments

  • Label with concentration, date, and buffer composition

• Reconstitution of lyophilized protein:

  • Centrifuge the vial briefly before opening to bring contents to the bottom

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

  • Allow complete dissolution before use, avoiding vigorous mixing that might denature the protein

• Critical handling precautions:

  • Repeated freeze-thaw cycles must be strictly avoided

  • Maintain cold chain during all handling steps

  • Use low-protein binding tubes and pipette tips to minimize loss during transfers

Following these methodological guidelines will maximize protein stability and experimental reproducibility when working with Mflv_3127.

How can advanced biophysical techniques enhance understanding of Mflv_3127 dynamics?

Understanding membrane protein dynamics is crucial for elucidating function, as these proteins often undergo conformational changes during their activity cycles . For Mflv_3127, several advanced biophysical techniques can provide insights into these dynamics:

• Single-molecule Förster resonance energy transfer (smFRET):

  • Strategically place fluorophore pairs at key positions in Mflv_3127

  • Monitor real-time conformational changes under various conditions

  • Can detect multiple conformational states not observable in ensemble measurements

• Molecular dynamics (MD) simulations:

  • Simulate Mflv_3127 behavior in a lipid bilayer environment

  • Predict conformational flexibility and potential binding sites

  • Identify water molecules or ions that might be important for function

• Hydrogen/deuterium exchange mass spectrometry (HDX-MS):

  • Map regions of the protein that become more or less solvent-exposed under different conditions

  • Identify dynamic regions that might be involved in substrate binding or conformational changes

  • Compare dynamics in different lipid environments

• Electron paramagnetic resonance (EPR) spectroscopy:

  • Introduce spin labels at strategic positions in Mflv_3127

  • Measure distances between labeled sites in different functional states

  • Particularly valuable for membrane proteins that resist crystallization

• Solid-state NMR:

  • Study Mflv_3127 dynamics directly in a lipid bilayer

  • Determine local mobility and orientation of specific regions

  • Can provide atomic-level insights into protein-lipid interactions

These advanced methodological approaches can reveal how Mflv_3127 functions at the molecular level, connecting structure to dynamics and ultimately to biological function.

What cutting-edge applications might benefit from Mflv_3127 research?

Research on Mflv_3127 can contribute to several cutting-edge applications in biotechnology and fundamental science:

• Synthetic biology applications:

  • Engineering bacterial membrane systems with modified or optimized transport capabilities

  • Development of biosensors using membrane protein components

  • Creation of minimal cell systems requiring essential membrane functions

• Antibiotic development:

  • If homologs exist in pathogenic mycobacteria, Mflv_3127 research could identify novel drug targets

  • Understanding of mycobacterial membrane protein structure-function relationships can guide rational drug design

  • Development of compounds that specifically disrupt essential membrane protein functions

• Membrane protein methodology advancement:

  • Optimization of protocols for expression and purification of challenging membrane proteins

  • Development of new stabilization strategies applicable to other membrane proteins

  • Refinement of computational prediction methods for membrane protein structure and function

• Environmental biotechnology:

  • As M. gilvum is known for biodegradation capabilities, understanding its membrane transporters might enhance bioremediation applications

  • Engineered systems incorporating mycobacterial membrane proteins could facilitate pollutant detection or degradation

• Fundamental understanding of protein evolution:

  • Comparative studies of UPF0060 family proteins across species can illuminate evolutionary patterns in membrane protein families

  • Investigation of how conserved structural elements maintain function despite sequence divergence

These applications demonstrate how basic research on Mflv_3127 can contribute to both fundamental scientific understanding and practical biotechnological innovations.