Recombinant Escherichia coli Inner membrane protein ynjI (ynjI)

What is the predicted membrane topology of YnjI and how can it be experimentally verified?

The inner membrane protein YnjI in Escherichia coli (346 amino acids) is predicted to contain multiple transmembrane domains. Based on bioinformatic analyses similar to those used for other E. coli inner membrane proteins, YnjI likely contains hydrophobic transmembrane segments that anchor the protein within the lipid bilayer.

Methodological approach for topology determination:

• Computational prediction: Begin with algorithms such as TMHMM, SOSUI, or TopPred to predict membrane-spanning regions. These tools analyze hydrophobicity patterns, charged residue distribution, and the positive-inside rule.

• PhoA/LacZ fusion approach: Generate systematic fusions of the alkaline phosphatase (PhoA) or β-galactosidase (LacZ) reporter enzymes at various positions throughout YnjI. PhoA is only active when located in the periplasm, while LacZ is only active in the cytoplasm. By measuring the enzymatic activity of different fusion constructs, you can map regions that face either side of the membrane .

• Cysteine scanning mutagenesis: Introduce cysteine residues at specific positions and test their accessibility to membrane-impermeable sulfhydryl reagents, providing information about exposed regions.

• C-terminal tagging strategy: As demonstrated for other E. coli inner membrane proteins, determining the location of YnjI's C-terminus (cytoplasmic or periplasmic) can significantly improve topology model accuracy .

For reliable experimental topology mapping of YnjI, combining computational predictions with at least two different experimental approaches is recommended for cross-validation.

What expression systems are most suitable for producing recombinant YnjI protein?

Producing functional recombinant inner membrane proteins like YnjI requires careful consideration of expression systems that accommodate membrane insertion and proper folding.

Recommended expression strategies:

• E. coli-based expression systems:

  • pET vector system with BL21(DE3) cells allows tunable expression using IPTG induction

  • pBAD system offering arabinose-inducible, titratable expression for better control of potential toxic effects

  • Low-temperature induction (16-25°C) to slow protein production and facilitate proper folding and membrane insertion

• Alternative host considerations:

  • C41(DE3) and C43(DE3) E. coli strains specifically engineered for membrane protein expression

  • Yeast systems (Pichia pastoris) for cases where E. coli-produced YnjI misfolding occurs

  • Baculovirus-infected insect cells for higher eukaryotic-like membrane environment

• Fusion tag strategies:

  • N-terminal His6-tag for purification while leaving C-terminal region unmodified

  • Maltose-binding protein (MBP) fusion to enhance solubility

  • Avi-tag biotinylation system for detection and immobilization applications

When designing expression constructs for YnjI, it's critical to consider that overexpression of membrane proteins can be toxic to host cells, so tightly regulated, moderate expression levels often yield better results than maximal production approaches.

How can the functional role of YnjI be investigated in the context of bacterial cell envelope biology?

Despite limited published information specifically on YnjI's function, systematic approaches can be employed to characterize its role within the bacterial cell envelope.

Investigative methodology:

• Genetic approaches:

  • Generate a conditional knockout strain (if YnjI is essential) or deletion mutant (if non-essential)

  • Perform complementation studies with wild-type and mutated versions

  • Analyze phenotypic changes under various stress conditions (temperature, pH, osmotic stress, antibiotics)

  • Create chimeric proteins with other characterized membrane proteins to identify functional domains

• Proteomic interactome mapping:

  • Apply protein-correlation-profiling (PCP) using SILAC labeling and peptidisc stabilization to identify interaction partners

  • Perform co-immunoprecipitation experiments with tagged YnjI followed by mass spectrometry

  • Use bacterial two-hybrid systems to screen for specific protein-protein interactions

  • Apply chemical cross-linking approaches to capture transient interactions

• Physiological assays:

  • Membrane integrity tests (sensitivity to detergents, antibiotics)

  • Membrane potential measurements

  • Assessment of osmotic stress responses

  • Analysis of lipid composition changes in YnjI mutants

• Comparative analysis:

  • Examine the function of homologous proteins in related bacterial species

  • Analyze expression patterns under different growth conditions and stress responses

  • Study co-expression patterns with functionally characterized genes

Given that other inner membrane proteins like YejM have been shown to play roles in cell envelope integrity and lipopolysaccharide assembly , investigating YnjI within these biological contexts may provide valuable insights into its function.

What purification strategies are most effective for isolating recombinant YnjI while maintaining its native structure?

Purifying inner membrane proteins presents unique challenges due to their hydrophobicity and requirement for a lipid environment to maintain native structure.

Recommended purification workflow:

• Membrane isolation and solubilization:

  • Isolate E. coli membranes by differential centrifugation after mechanical or enzymatic cell disruption

  • Test multiple detergents for efficient solubilization:

    • Mild detergents like DDM (n-dodecyl-β-D-maltoside) or LDAO (lauryldimethylamine oxide)

    • Comparative solubilization efficiency test with DDM, LDAO, β-OG, and DOC as demonstrated for other membrane proteins

  • Optimize detergent concentration and solubilization time (typically 15-30 minutes at 4°C)

• Affinity chromatography:

  • Ni-NTA purification for His-tagged YnjI constructs

  • Include appropriate detergent in all buffers (typically 0.02-0.05% DDM for wash and elution steps)

  • Add glycerol (10%) to enhance protein stability

  • Consider on-column detergent exchange if necessary

• Size exclusion chromatography:

  • Further purify by SEC in buffer containing suitable detergent

  • Analyze oligomeric state and protein-detergent complex size

• Alternative membrane mimetics:

  • Consider transferring purified YnjI from detergent to more native-like environments:

    • Reconstitution into peptidiscs (using NSPr peptide) which has shown superior results for maintaining membrane protein complexes compared to SMALPs

    • Incorporation into nanodiscs with defined lipid composition

    • Liposome reconstitution for functional studies

• Quality control:

  • SDS-PAGE analysis with Coomassie staining

  • Western blotting with anti-His antibody or custom YnjI antibodies

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to assess secondary structure integrity

The peptidisc approach has demonstrated particular effectiveness for maintaining membrane protein complexes and interactions that may be disrupted in detergent , potentially making it valuable for YnjI structural and functional studies.

What experimental approaches can be used to study potential interactions between YnjI and other membrane or cytosolic proteins?

Investigating the protein interaction network of YnjI requires specialized techniques adapted for membrane proteins.

Methodological strategies:

• In vivo crosslinking and co-immunoprecipitation:

  • Use membrane-permeable crosslinkers like formaldehyde or DSP (dithiobis(succinimidyl propionate))

  • Perform immunoprecipitation with anti-YnjI antibodies or via affinity tags

  • Identify interaction partners by mass spectrometry

  • Validate interactions with reciprocal pull-downs

• Peptidisc-based interactome mapping:

  • Apply the SEC-PCP-SILAC workflow as described for E. coli membrane proteins :

    • Solubilize membranes with mild detergent (preferably DDM)

    • Reconstitute the membrane proteome into peptidiscs

    • Fractionate by size-exclusion chromatography

    • Compare elution profiles of YnjI with potential interaction partners

  • This approach has successfully identified previously undetected interactions between Sec and Bam complexes

• Bacterial two-hybrid systems:

  • Adapt BACTH (Bacterial Adenylate Cyclase Two-Hybrid) for membrane protein interaction studies

  • Test YnjI against candidate partners such as other inner membrane proteins or components of membrane protein complexes

  • Design truncated versions of YnjI containing specific domains to map interaction regions

• Fluorescence-based approaches:

  • FRET (Förster Resonance Energy Transfer) using fluorescent protein fusions

  • Split-GFP complementation assays to visualize interactions in vivo

  • Bimolecular Fluorescence Complementation (BiFC) to detect protein complexes

• Mass spectrometry techniques:

  • SILAC-based comparative AP-MS (Affinity Purification-Mass Spectrometry)

  • Label-free quantification to detect enriched interaction partners

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces

The peptidisc-based approaches have shown particular value for preserving membrane protein interactions that are often lost in traditional detergent-based methods , potentially revealing YnjI interactions that would otherwise remain undetected.

How does YnjI expression change in response to different environmental stresses and growth conditions?

Understanding the regulation of YnjI expression can provide insights into its physiological role.

Experimental approaches:

• Transcriptional analysis:

  • RT-qPCR to quantify ynjI mRNA levels under various conditions:

    • Growth phase (exponential vs. stationary phase)

    • Nutrient limitation

    • pH stress

    • Osmotic stress

    • Temperature shifts

    • Antibiotic exposure

  • RNA-seq for genome-wide expression analysis to identify co-regulated genes

  • Promoter-reporter fusion (luciferase or GFP) to monitor expression dynamics in real-time

• Proteomic quantification:

  • Western blotting with YnjI-specific antibodies

  • SILAC-based quantitative proteomics to compare expression levels across conditions

  • Targeted mass spectrometry (SRM/MRM) for absolute quantification

• Regulatory element identification:

  • In silico analysis of the ynjI promoter region for transcription factor binding sites

  • Chromatin immunoprecipitation (ChIP) to identify proteins binding to the ynjI promoter

  • Systematic promoter mutations to identify critical regulatory elements

• Comparison with other stress-responsive membrane proteins:

  • Compare expression patterns with known stress-responsive inner membrane proteins like YqjD, which is regulated by sigma factor RpoS and expressed during stationary phase

  • Determine if YnjI shows similar RpoS-dependent regulation

  • Examine if YnjI is co-regulated with other cell envelope stress response genes

Based on patterns observed in other inner membrane proteins like YqjD , it would be valuable to specifically examine YnjI expression during the transition from exponential to stationary phase and under various cellular stress conditions to understand its potential role in stress adaptation.

What structural characterization methods are suitable for studying YnjI?

Structural studies of membrane proteins require specialized approaches due to their hydrophobic nature and requirement for lipid environments.

Methodological approaches:

Given that structures for other E. coli inner membrane proteins like YejM have been successfully determined (e.g., YejM's periplasmic domain at 2.35 Å resolution ), similar approaches could be adapted for YnjI structural characterization.

How can genetic engineering approaches be used to investigate YnjI function?

Genetic manipulation strategies provide powerful tools for investigating YnjI's role in bacterial physiology.

Genetic engineering approaches:

• Generation of conditional mutants:

  • Create depletion strains where ynjI expression is controlled by inducible promoters

  • Develop temperature-sensitive alleles through random or site-directed mutagenesis

  • Utilize degron-based systems for controlled protein degradation

• Domain mapping and functional analysis:

  • Generate systematic truncations to identify functional domains

  • Create chimeric proteins with homologous or functionally related proteins

  • Perform alanine-scanning mutagenesis of conserved residues

  • Design point mutations based on predicted functionally important sites

• High-throughput screens:

  • Perform synthetic genetic array (SGA) analysis to identify genetic interactions

  • Apply Tn-seq approaches to identify genes that become essential in a ΔynjI background

  • Screen for suppressors of ynjI mutant phenotypes

• In vivo localization studies:

  • Create fluorescent protein fusions to determine subcellular localization

  • Design split-GFP constructs to verify membrane topology

  • Perform time-lapse microscopy to track dynamic changes in localization

• CRISPR/Cas9-based approaches:

  • Generate precise genomic modifications without leaving selection markers

  • Create libraries of guide RNAs targeting different regions of ynjI for functional screening

  • Employ CRISPRi for tunable repression of ynjI expression

• Complementation studies:

  • Test if YnjI homologs from other bacteria can complement E. coli ynjI mutants

  • Determine if paralogs with similar structure can substitute functionally

These approaches can be particularly informative when integrated with phenotypic analyses to determine how YnjI contributes to cell envelope integrity, stress responses, and other cellular processes, similar to studies conducted on other inner membrane proteins like YejM .

How does YnjI compare to other characterized inner membrane proteins in E. coli, and what can be inferred about its function?

Comparative analysis with well-characterized inner membrane proteins can provide valuable insights into potential YnjI functions.

Comparative analysis approach:

• Sequence homology and domain architecture:

  • Perform sequence alignment with characterized inner membrane proteins

  • Identify conserved domains and motifs that may suggest functional similarities

  • Compare with proteins like:

    • YejM (involved in cell envelope synthesis and outer membrane modifications)

    • YqjD (stationary phase-induced, ribosome-associated inner membrane protein)

    • YhiM (involved in copper homeostasis and envelope stress response)

• Expression pattern comparison:

  • Analyze if YnjI expression correlates with specific growth phases like YqjD (stationary phase)

  • Determine if YnjI is regulated by specific stress response systems like YhiM (CpxAR system)

  • Examine co-expression patterns with functionally related proteins

• Phenotypic comparison of mutants:

  • Compare phenotypes of ynjI mutants with those of other inner membrane proteins:

    • Membrane permeability defects (like yejM mutants)

    • Temperature sensitivity (observed in yejM mutants)

    • Growth inhibition effects (seen with YqjD overexpression)

    • Metal ion sensitivity (as in yhiM mutants)

• Protein interaction network comparison:

  • Compare YnjI's interactome with that of other membrane proteins using techniques like peptidisc-based SEC-PCP-SILAC

  • Identify shared interaction partners that may suggest functional pathways

• Evolutionary conservation analysis:

  • Determine if YnjI is conserved across bacterial species or specific to certain phylogenetic groups

  • Compare with proteins like YqjD which has limited distribution even among closely related bacteria

Based on the limited information available specifically for YnjI, comparative studies with better-characterized proteins like YejM, YqjD, and YhiM represent a valuable approach to generating testable hypotheses about its function in bacterial physiology.

What functional assays can be developed to characterize the biochemical activities of purified YnjI?

Developing biochemical assays for inner membrane proteins requires considering potential activities based on sequence features and cellular localization.

Functional assay development strategy:

• Transporter/channel activity assessment:

  • Liposome reconstitution followed by:

    • Ion flux measurements using fluorescent dyes

    • Substrate transport assays with radiolabeled compounds

    • Electrophysiological measurements (planar lipid bilayer or patch-clamp)

  • Whole-cell transport assays comparing wild-type and ΔynjI strains

• Enzymatic activity screening:

  • Based on the example of YejM (identified as a metalloenzyme with phosphatase activity) , test for:

    • Phosphatase activity with various substrates

    • Hydrolase activity against lipid substrates

    • Metal ion binding and metalloenzyme activity

  • Screen with substrate libraries to identify potential enzymatic functions

• Protein-protein interaction assays:

  • Surface plasmon resonance (SPR) with immobilized YnjI

  • Microscale thermophoresis (MST) to measure binding affinities

  • Pull-down assays with potential interaction partners identified through proteomic approaches

  • Biolayer interferometry with purified candidate interactors

• Lipid interaction studies:

  • Lipid binding assays using:

    • Fluorescently labeled lipids

    • Liposome flotation assays

    • Monolayer insertion experiments

  • Test for specific lipid preferences or modifications similar to YejM's role in outer membrane lipid composition

• Structural changes in response to conditions:

  • Monitor conformational changes using:

    • Intrinsic tryptophan fluorescence

    • Environmentally sensitive fluorescent probes

    • Hydrogen-deuterium exchange mass spectrometry

  • Test responses to membrane potential, pH, or ion gradients

Considering that other inner membrane proteins like YejM have been found to possess unexpected enzymatic activities (phosphatase activity) , a broad initial screening approach for YnjI is recommended, followed by focused characterization of identified activities.

How might YnjI be involved in bacterial stress responses and adaptation?

Many inner membrane proteins play crucial roles in sensing and responding to environmental stresses, suggesting potential roles for YnjI.

Investigative framework:

• Stress sensitivity phenotyping:

  • Compare growth and survival of wild-type and ΔynjI strains under:

    • Osmotic stress (high/low osmolarity) similar to testing done for YciB

    • Temperature stress (heat/cold shock)

    • pH stress (acidic/alkaline conditions)

    • Oxidative stress (H₂O₂, paraquat)

    • Envelope stress (detergents, antibiotics targeting cell envelope)

    • Metal ion stress (copper, zinc, iron limitation/excess)

• Gene expression analysis during stress:

  • Monitor ynjI expression under various stress conditions using:

    • RT-qPCR

    • Promoter-reporter fusions

    • RNA-seq

  • Compare with known stress-responsive genes to identify potential regulatory networks

• Transcriptional regulation studies:

  • Determine if ynjI is regulated by stress-responsive transcription factors:

    • RpoS (general stress response) as observed for YqjD

    • CpxR (envelope stress) as observed for YhiM

    • OmpR (osmotic stress)

    • Others (SoxS, OxyR, Fur, etc.)

• Protein localization during stress:

  • Track localization of fluorescently tagged YnjI under normal and stress conditions

  • Determine if its distribution changes during adaptation to stress

• Interaction partner dynamics:

  • Identify stress-dependent changes in YnjI's interaction network

  • Determine if YnjI associates with stress-response complexes under specific conditions

Based on findings for other inner membrane proteins like YciB (involved in osmotic stress responses) , YqjD (stationary phase-induced, RpoS-regulated) , and YhiM (copper stress, CpxAR system) , investigating YnjI's role in various stress responses represents a promising direction for functional characterization.

What approaches can be used to determine if YnjI is essential for E. coli viability and growth?

Determining essentiality requires careful genetic manipulation and conditional expression systems.

Methodological approaches:

• Deletion strain construction attempts:

  • Apply standard gene replacement techniques to attempt complete deletion

  • Use λ-Red recombinase-mediated gene replacement as used for other E. coli genes

  • If deletion is lethal, this suggests essentiality (as observed for full yejM gene)

• Conditional expression systems:

  • Generate strains where chromosomal ynjI is deleted but complemented by:

    • Plasmid-borne ynjI under an inducible promoter (arabinose, IPTG, tetracycline)

    • Integrated copy with controllable expression

  • Test growth dependency on inducer presence/concentration

• Depletion experiments:

  • Design strains expressing YnjI under tight regulatory control

  • Monitor physiological consequences of YnjI depletion over time

  • Analyze morphological changes, growth rates, and viability

• Temperature-sensitive alleles:

  • Generate temperature-sensitive mutations in ynjI

  • Characterize growth at permissive vs. non-permissive temperatures

  • Analyze phenotypic consequences of loss of function

• CRISPR interference (CRISPRi):

  • Design guide RNAs targeting ynjI

  • Use dCas9-based transcriptional repression

  • Titrate repression levels to determine minimum expression requirements

• Transposon insertion analysis:

  • Examine existing Tn-seq datasets for insertion frequency in ynjI

  • Low insertion frequency would suggest essentiality

  • Conduct new Tn-seq experiments under various conditions to determine condition-dependent essentiality

• Suppressor screening:

  • If YnjI is essential, screen for suppressor mutations that allow growth in its absence

  • Identify alternative pathways or bypass mechanisms

  • Look for specific overexpression suppressors (similar to AcpT suppression of YejM deficiency)

The approach used for YejM, where truncated forms were viable while complete deletion was lethal , provides a valuable model for investigating YnjI essentiality and potentially identifying critical domains.

How can researchers investigate potential roles of YnjI in bacterial pathogenesis and virulence?

Even though YnjI is found in laboratory E. coli strains, understanding its potential contribution to pathogenic E. coli virulence may provide valuable insights.

Research strategy:

• Comparative genomics approach:

  • Compare ynjI sequence and conservation between commensal and pathogenic E. coli strains

  • Identify any pathogen-specific variations in sequence or expression regulation

  • Examine presence/absence and sequence conservation in other pathogenic bacteria

• Infection model studies:

  • Generate ynjI deletion or depletion strains in pathogenic E. coli backgrounds

  • Test virulence in appropriate infection models:

    • Cell culture invasion/adhesion assays

    • Galleria mellonella infection model

    • Mouse infection models for appropriate pathotypes

  • Measure competitive index of wild-type vs. mutant in mixed infections

• Host-pathogen interaction studies:

  • Investigate if YnjI affects:

    • Resistance to host antimicrobial peptides

    • Biofilm formation capacity

    • Intracellular survival

    • Resistance to oxidative burst

• Virulence factor expression analysis:

  • Determine if YnjI deletion affects expression of known virulence factors

  • Assess if stress response pathways linked to virulence are impacted

• Antibiotic resistance contribution:

  • Test if YnjI affects:

    • Minimum inhibitory concentrations of different antibiotics

    • Membrane permeability to antibiotics

    • Persistence formation under antibiotic stress

• In vivo expression studies:

  • Use techniques like IVET (In Vivo Expression Technology) to determine if ynjI is specifically upregulated during infection

  • Create reporter strains to monitor ynjI expression during various stages of infection

Given that other inner membrane proteins like YejM have been linked to cell envelope integrity and potentially antibiotic resistance , investigating YnjI's contribution to pathogen survival in host environments and response to antimicrobial challenges would be particularly valuable.

What is the evolutionary conservation of YnjI across bacterial species and what can this tell us about its function?

Evolutionary analysis can provide significant insights into protein function based on patterns of conservation.

Evolutionary analysis methodology:

• Phylogenetic distribution mapping:

  • Conduct BLAST searches against bacterial genomes

  • Determine presence/absence patterns across bacterial phyla

  • Create phylogenetic tree of YnjI homologs

  • Compare distribution with functionally characterized homologs

• Sequence conservation analysis:

  • Perform multiple sequence alignment of YnjI homologs

  • Identify:

    • Universally conserved residues (likely critical for function)

    • Clade-specific conservation patterns

    • Rapidly evolving regions

  • Map conservation onto predicted structural models

• Synteny analysis:

  • Examine genomic context of ynjI across species

  • Identify conserved gene neighborhoods that may suggest functional associations

  • Look for co-evolution with specific metabolic or stress response pathways

• Domain architecture comparison:

  • Identify variation in domain organization across species

  • Detect domain fusion/fission events that may link to other functional domains

  • Compare with paralogs in the same species (if any)

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify regions under purifying or positive selection

  • Detect signatures of selection that might indicate adaptation to specific niches

• Comparative functional genomics:

  • Leverage existing mutant phenotype data across species

  • Integrate with gene expression data from multiple species

  • Identify consistent phenotypic associations across evolutionary distance

This approach could reveal whether YnjI belongs to a broadly distributed protein family (like YejM) or has a more restricted distribution (like YqjD, which was primarily found in E. coli and closely related Shigella ), providing context for functional hypotheses.

How might post-translational modifications affect YnjI function?

Post-translational modifications (PTMs) often regulate membrane protein function and stability.

Investigation approaches:

• PTM identification:

  • Apply mass spectrometry-based proteomic approaches:

    • Enrichment strategies for specific modifications (phosphorylation, glycosylation)

    • Multiple fragmentation methods (CID, ETD, HCD) for comprehensive coverage

    • Targeted MS/MS for suspected modification sites

  • Analyze purified YnjI from different growth conditions to detect condition-specific modifications

• Functional impact assessment:

  • Generate site-directed mutants that:

    • Mimic modifications (phosphomimetic mutations: S/T→D/E)

    • Prevent modifications (S/T→A, K→R, etc.)

  • Test functional consequences in vivo and in vitro

  • Assess impact on protein-protein interactions and activity

• Regulatory enzyme identification:

  • Screen for kinases/phosphatases that modify YnjI

  • Identify other enzymes responsible for detected modifications

  • Determine environmental signals that trigger modification

• Lipid modifications:

  • Investigate potential lipid modifications common in membrane proteins:

    • Palmitoylation

    • Myristoylation

    • Prenylation

  • Assess impact on membrane localization and protein-lipid interactions

• Proteolytic processing:

  • Determine if YnjI undergoes regulated proteolysis

  • Identify protease cleavage sites

  • Assess functional consequences of processing

Given that other inner membrane proteins like YejM have been found to undergo functionally significant modifications (YejM can be phosphorylated, affecting its function) , investigating potential PTMs of YnjI represents an important avenue for understanding its regulation and activity.