Recombinant Probable protein-export membrane protein secG (secG)

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Description

Introduction to Recombinant Probable Protein-Export Membrane Protein SecG (secG)

Recombinant Probable protein-export membrane protein SecG (secG) is a genetically engineered variant of the bacterial SecG protein, produced through heterologous expression systems. This membrane protein is a component of the SecYEG translocon, a conserved machinery essential for transporting proteins across bacterial inner membranes and inserting membrane proteins into lipid bilayers . While SecG is not universally essential for viability, its recombinant form is studied for its role in enhancing translocation efficiency and understanding auxiliary functions in protein export .

Molecular Architecture

SecG consists of two transmembrane domains (TMs) connected by a cytosolic loop . In its recombinant form, partial sequences are often utilized (e.g., Uniprot: P66792) . Structural studies indicate that SecG interacts with SecY and SecE to stabilize the translocon’s conformation, particularly in dynamic states during protein transport .

Role in the SecYEG Translocon

FunctionMechanismSource
Stabilizes transloconPrevents degradation of SecY in the absence of SecE
Enhances translocationStimulates ATPase activity of SecA (12-fold increase)
Modulates membrane dynamicsRegulates phospholipid metabolism under stress (e.g., cold sensitivity)

Production Parameters

Recombinant SecG is typically expressed in mammalian cell systems (e.g., Mycobacterium bovis) and purified to >85% purity (SDS-PAGE) . Key specifications include:

AttributeValue
Product CodeCSB-MP353626MVH1
Source OrganismMycobacterium bovis (strain ATCC BAA-935 / AF2122/97)
Storage-20°C/-80°C (lyophilized: 12 months; liquid: 6 months)
ReconstitutionDeionized sterile water (0.1–1.0 mg/mL with 5–50% glycerol)

Research Applications

  • Translocation Assays: Reconstituted SecYEG complexes (including SecG) support protease-protected translocation of proOmpA in vitro .

  • ATPase Activity Studies: SecA’s ATPase activity is stimulated 12-fold in the presence of SecYEG .

  • Phospholipid Metabolism: SecG’s deletion exacerbates cold sensitivity linked to glycerol-3-phosphate depletion, highlighting its role in membrane stability .

Auxiliary Role in Export

Deletion of secG in E. coli does not universally cause lethality but reduces export efficiency, particularly under stress (e.g., cold) . This auxili-ary function is strain-dependent and influenced by secondary mutations (e.g., glpR) that disrupt phospholipid homeostasis .

Strain-Dependent Phenotypes

ConditionPhenotypeMechanism
ΔsecG (wild-type)Mild export defect, no cold sensitivityCompensated by other pathways
ΔsecG + glpR mutationCold-sensitive, impaired phospholipid synthesisGlycerol-3-phosphate depletion

Interaction with SecY and SecE

SecG’s absence destabilizes SecY, leading to degradation by FtsH protease . This underscores SecG’s role in maintaining translocon integrity, particularly during high-flux transport .

Product Specs

Form
Lyophilized powder
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate specific format requirements. Please indicate your preference in the order notes, and we will prepare accordingly.
Lead Time
Delivery time may vary depending on the purchasing method and location. Please consult your local distributors for specific delivery estimates.
Note: All our proteins are shipped with standard blue ice packs by default. If dry ice shipping is required, please inform us in advance as additional charges may apply.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend centrifuging the vial briefly before opening to ensure the contents settle at the bottom. Reconstitute the protein in deionized sterile water to a concentration between 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard final glycerol concentration is 50%, which can be used as a reference.
Shelf Life
The shelf life is influenced by various factors including storage conditions, buffer ingredients, temperature, and the protein's inherent stability.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type will be determined during the manufacturing process.
The tag type will be determined during the production process. If you have a specific tag type in mind, please inform us and we will prioritize its development.
Synonyms
secG; ycf47; Probable protein-export membrane protein secG
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-71
Protein Length
full length protein
Species
Porphyra purpurea (Red seaweed) (Ulva purpurea)
Target Names
secG
Target Protein Sequence
MEQILKFLWYISTIILVFSILIHNPKSEGLGTIGSQNQFFSNTRSTENTLNKVTWLFLAL FLLFTTILAIN
Uniprot No.

Target Background

Function
Involved in protein export. Participates in an early event of protein translocation across the chloroplast thylakoid membrane (Potential).
Protein Families
SecG family
Subcellular Location
Plastid, chloroplast thylakoid membrane; Multi-pass membrane protein.

Q&A

What is the structure and function of SecG within the SecYEG translocon?

SecG functions as an integral component of the SecYEG translocon, which serves as the main conduit for protein translocation across the bacterial cytoplasmic membrane. As part of this complex, SecG works in conjunction with SecY and SecE to form a channel through which unfolded proteins can pass. The SecYEG complex interacts with the SecA ATPase to facilitate the energy-dependent translocation process . Structurally, SecG is positioned within the complex in a manner that allows it to undergo topological inversion during protein translocation, which is believed to enhance the efficiency of the process by facilitating the insertion and deinsertion of the SecA protein.

How does SecG contribute to protein translocation efficiency?

SecG enhances the efficiency of protein translocation through several mechanisms:

  • Topological inversion: SecG can undergo a unique topological inversion during the translocation process

  • SecA interaction: SecG facilitates the membrane insertion and deinsertion of SecA

  • Stabilization function: SecG helps stabilize the SecYE complex in the membrane

  • PMF utilization: While SecG itself is not directly involved in proton movement, it contributes to the efficiency of PMF-dependent translocation

These functions, while not absolutely essential for protein translocation in all conditions, become particularly important under stressful conditions or at lower temperatures where membrane fluidity is reduced .

What experimental systems are commonly used to study recombinant SecG?

Several experimental systems have been developed for studying recombinant SecG:

Experimental SystemAdvantagesLimitations
E. coli expression systemsHigh yield, genetic tractability, native environmentPotential toxicity when overexpressed
T. thermophilus systemsThermostability, crystallization potentialLess physiologically relevant to mesophilic organisms
Liposome reconstitutionControlled lipid environment, isolated system analysisLacks cellular components that may influence function
In vitro translation systemsDirect analysis of translocationLimited in reproducing all aspects of cellular environment

The selection of an appropriate system depends on the specific research questions being addressed and the experimental techniques to be employed.

How does the ATP-independent, PMF-driven protein translocation mechanism relate to SecG function?

While SecG itself is not directly responsible for PMF utilization, it contributes to the efficiency of PMF-dependent translocation steps. Research has identified that protein translocation across bacterial membranes includes both ATP-dependent (via SecA) and ATP-independent (PMF-driven) components. The post-initiation mode of translocation can occur in the absence of ATP and SecA, relying instead on PMF and membrane-integrated components like SecDF .

The relationship between SecG and this ATP-independent mechanism involves:

  • Conformational support: SecG may assist in maintaining optimal conformation of the translocon for PMF utilization

  • Interaction with SecDF: SecG potentially coordinates with SecDF, which has been shown to function as a membrane-integrated chaperone powered by PMF

  • Channel stability: SecG contributes to the stability of the translocation channel during PMF-driven movement of the substrate

Studies using electrophysiological analyses have revealed that associated components like SecDF conduct protons in a pH- and unfolded protein-dependent fashion, suggesting a complex interplay between the various Sec components during translocation .

What are the current challenges in studying the real-time dynamics of SecG conformational changes?

The study of real-time SecG conformational changes presents several significant challenges:

  • Temporal resolution limitations: Conventional structural methods (X-ray crystallography, cryo-EM) provide static snapshots rather than dynamic information

  • Membrane environment complexity: Maintaining native membrane environment while enabling observation of conformational changes

  • Transient interactions: The rapid and transient nature of SecG topological inversions

  • Coordinated movements: Distinguishing SecG movements from those of other Sec components

Current advanced approaches to address these challenges include:

  • Single-molecule FRET to track distance changes between strategically placed fluorophores

  • Site-directed spin labeling combined with EPR spectroscopy

  • Hydrogen-deuterium exchange mass spectrometry to monitor solvent accessibility changes

  • Molecular dynamics simulations to predict conformational transitions

These techniques must be combined with careful experimental design to overcome the inherent difficulties in membrane protein research.

How do mutations in conserved regions of SecG affect its interaction with other Sec components?

Mutations in conserved regions of SecG can significantly impact its interactions with other Sec components, particularly SecY, SecE, and SecA. Research indicates that:

  • Interface mutations: Alterations at the SecG-SecY interface can disrupt the stability of the entire complex

  • Topological inversion domains: Mutations in regions involved in topological inversion can prevent conformational changes

  • SecA interaction sites: Modifications to SecA interaction domains can reduce translocation efficiency

Analogous to findings with SecDF, where conserved Asp and Arg residues at transmembrane interfaces are crucial for function , SecG likely contains similar conserved residues essential for its proper function within the complex. Complementation tests similar to those performed for SecDF mutations can reveal the functional significance of these conserved regions in SecG.

What approaches can be used to optimize expression and purification of recombinant SecG?

The optimization of recombinant SecG expression and purification requires specialized approaches due to its membrane protein nature:

StepMethodologyConsiderations
Expression vectorpET-based systems with tunable promotersOptimize induction conditions to prevent toxicity
Host strainC43(DE3), C41(DE3), or BL21(DE3)pLysSStrains designed for membrane protein expression
InductionLow temperature (16-20°C), reduced IPTG concentrationSlow expression to allow proper membrane insertion
SolubilizationDetergent screening (DDM, LDAO, FC-12)Select detergents that maintain native structure
PurificationIMAC followed by size exclusion chromatographyRemove detergent micelles and aggregates
Quality controlCircular dichroism, thermal shift assaysVerify proper folding and stability

The expression and purification process should include controls to verify that the recombinant SecG retains its native structure and function, possibly through complementation assays in SecG-deficient strains or in vitro translocation assays.

How can researchers design experiments to study SecG-dependent protein translocation in vitro?

Designing robust in vitro translocation assays for SecG-dependent processes requires:

  • Reconstitution system preparation:

    • Purification of SecYEG components (including SecG)

    • Preparation of model preprotein substrates (e.g., proOmpA)

    • Isolation of SecA and other necessary factors

    • Generation of inverted membrane vesicles or proteoliposomes

  • Translocation assay design:

    • Establishment of energy sources (ATP and/or PMF)

    • Methods for generating and measuring PMF in vitro

    • Protection assays to measure translocation efficiency

    • Real-time fluorescence-based translocation monitoring

  • Experimental variables to consider:

    • Temperature effects (particularly important for SecG function)

    • pH gradients and their influence on translocation

    • Lipid composition of membranes

    • Presence/absence of additional factors (SecDF, YidC)

  • Controls and comparisons:

    • SecG-depleted systems to assess specific contributions

    • Mutant variants to identify critical residues

    • Comparison of ATP-dependent vs. PMF-dependent steps

Similar to the approaches used for studying SecDF's PMF utilization , researchers should consider electrophysiological methods such as inside-out patch-clamp experiments to analyze ion conductance properties and their relationship to protein translocation.

What structural analysis techniques are most suitable for characterizing SecG interactions?

Multiple structural analysis techniques can be employed to characterize SecG interactions within the translocon complex:

  • X-ray crystallography:

    • Advantages: High-resolution structural information

    • Challenges: Difficulty in crystallizing membrane protein complexes

    • Adaptations: Lipidic cubic phase crystallization, antibody fragment co-crystallization

  • Cryo-electron microscopy:

    • Advantages: No crystallization requirement, visualization of different conformational states

    • Challenges: Sample preparation, achieving high resolution with small membrane proteins

    • Recent advances: Direct electron detectors and computational classification methods

  • Cross-linking mass spectrometry:

    • Advantages: Identification of specific interaction sites, applicable in native conditions

    • Methodology: Photo-activatable or chemical crosslinkers followed by MS analysis

    • Data analysis: Specialized software to identify crosslinked peptides and map interaction sites

  • NMR spectroscopy:

    • Applications: Dynamics studies, residue-specific interactions

    • Limitations: Size constraints, complex spectral interpretation

    • Selective labeling strategies to focus on specific interactions

These techniques can be used complementarily to develop a comprehensive understanding of SecG's structural interactions, similar to the multi-method approaches used to elucidate the structure and function of SecDF .

How should researchers interpret conflicting results between in vivo and in vitro studies of SecG function?

Conflicting results between in vivo and in vitro studies of SecG function require careful interpretation:

  • Systematic analysis of differences:

    • Environmental factors (membrane composition, crowding effects)

    • Concentration differences between reconstituted and native systems

    • Presence/absence of accessory factors in different experimental setups

    • Post-translational modifications not reproduced in vitro

  • Reconciliation approaches:

    • Progressive complexity models (from purified components to cell extracts)

    • Correlation with physiological conditions (temperature, pH, ionic strength)

    • Genetic complementation studies to validate in vitro findings

    • Quantitative comparisons of kinetic parameters across systems

  • Interpretation framework:

    • Distinguish essential vs. enhancing roles under different conditions

    • Consider conditional essentiality (stress, temperature sensitivity)

    • Evaluate evolutionary conservation to assess functional importance

    • Analyze synthetic phenotypes with other translocation component mutations

When evaluating data, researchers should consider that SecG may function primarily as an efficiency-enhancing component rather than an absolutely essential one, similar to observations about SecDF's role in enhancing protein translocation through PMF utilization .

What statistical approaches are appropriate for analyzing SecG mutant phenotypes?

Appropriate statistical approaches for analyzing SecG mutant phenotypes include:

  • Quantitative growth analysis:

    • Comparing growth rates under various conditions

    • Statistical models: ANOVA with post-hoc tests for multiple comparisons

    • Growth curve analysis with non-linear regression models

  • Protein translocation efficiency measurements:

    • Pulse-chase assays with densitometric quantification

    • Statistical analysis: Paired t-tests or non-parametric equivalents

    • Multi-factor analysis to assess interactions between mutations and conditions

  • Structure-function correlation approaches:

    • Multiple sequence alignment conservation analysis

    • Correlation of mutation effects with structural features

    • Regression models to predict functional impacts from sequence/structural data

  • Comprehensive phenotypic analysis:

    • Principal component analysis to identify major phenotypic dimensions

    • Hierarchical clustering of mutants based on phenotypic similarities

    • Bayesian networks to model causal relationships between genetic changes and phenotypes

The statistical approaches should be selected based on the specific experimental design, data distribution, and research questions, with appropriate attention to sample sizes, biological replicates, and potential confounding variables .

How can researchers effectively design simulations to study SecG dynamics?

Effective molecular dynamics simulation design for studying SecG dynamics requires:

  • System preparation considerations:

    • Accurate membrane composition reflecting bacterial inner membrane

    • Proper embedding of the SecYEG complex

    • Inclusion of key interacting molecules (SecA, preprotein substrates)

    • Appropriate water model and ion conditions

  • Simulation approaches:

    • Equilibrium MD to study native state fluctuations

    • Steered MD to investigate translocation processes

    • Umbrella sampling to determine energy landscapes of conformational changes

    • Coarse-grained models for longer timescale events

  • Analysis methods:

    • Principal component analysis to identify major modes of motion

    • Contact map analysis to track changing interactions

    • Channel dimension analysis to correlate with function

    • Correlation analysis to identify allosteric networks

  • Validation strategies:

    • Comparison with experimental observables (FRET distances, accessibility data)

    • Consistency across multiple force fields

    • Experimental testing of simulation-derived hypotheses

    • Convergence analysis to ensure adequate sampling

Successful simulation approaches should balance computational tractability with biological relevance, potentially focusing on specific aspects of SecG function such as its topological inversion or interaction with translocating substrates .

What emerging technologies show promise for advancing SecG research?

Several emerging technologies offer significant potential for advancing SecG research:

  • Single-molecule approaches:

    • High-speed AFM for real-time visualization of conformational changes

    • Nanodiscs combined with single-molecule spectroscopy

    • Zero-mode waveguides for studying translocation kinetics

    • Single-molecule force spectroscopy to measure energy landscapes

  • Advanced structural methods:

    • Time-resolved crystallography using XFEL sources

    • Integrative structural biology combining multiple data sources

    • Cryo-electron tomography of SecG in native membranes

    • Mass photometry for studying complex assembly dynamics

  • Genetic and genomic approaches:

    • CRISPR interference for tunable expression control

    • Deep mutational scanning to comprehensively map function

    • Ribosome profiling to study co-translational engagement with SecG

    • Synthetic biology approaches to engineer novel functionalities

  • Computational advances:

    • AI-accelerated molecular dynamics with enhanced sampling

    • Machine learning for predicting SecG substrate interactions

    • Quantum mechanics/molecular mechanics for studying catalytic effects

    • Network analysis approaches for understanding system-level impacts

These technologies, when applied appropriately, can address the current knowledge gaps regarding SecG function and dynamics .

How might cross-disciplinary approaches enhance our understanding of SecG function?

Cross-disciplinary approaches can significantly enhance SecG research through:

  • Biophysics-biochemistry integration:

    • Combining structural studies with functional assays

    • Correlating energy landscapes with biochemical kinetics

    • Linking membrane biophysics to protein function

    • Developing novel spectroscopic tools for membrane protein analysis

  • Systems biology perspectives:

    • Network analysis of Sec pathway components and substrates

    • Global impacts of SecG perturbation on proteome localization

    • Metabolic consequences of translocation efficiency changes

    • Evolutionary analysis of SecG across diverse bacterial species

  • Synthetic biology applications:

    • Engineering SecG variants with novel functionalities

    • Creating minimal translocation systems with defined components

    • Developing biosensors based on SecG conformational changes

    • Exploiting SecG for biotechnological protein production

  • Computational-experimental synergy:

    • Iterative refinement of models through experimental validation

    • Design of targeted experiments based on computational predictions

    • Development of quantitative models incorporating multiple data types

    • Machine learning approaches to interpret complex datasets

By integrating knowledge and methodologies across disciplines, researchers can develop more comprehensive models of SecG function within the broader context of bacterial protein export systems .

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