Recombinant Putative methyl-accepting chemotaxis protein yoaH (yoaH)

What expression systems are optimal for recombinant yoaH production while preserving post-translational modification capacity?

Recombinant yoaH requires expression systems capable of supporting methylation and CheB-dependent modifications critical for chemotaxis signaling . E. coli BL21(DE3) remains the default host due to its compatibility with endogenous methyltransferase systems , but solubility varies significantly across vector systems. For instance, pET-28a(+) vectors with N-terminal His-tags yield 15–20 mg/L of soluble yoaH in autoinduction media at 18°C, while cytoplasmic expression in Pichia pastoris GS115 using pPICZαA achieves 8–12 mg/L with enhanced glycosylation resistance . A comparative analysis of expression hosts is provided below:

For studies requiring CheB-mediated demethylation, E. coli ΔcheB knockout strains transformed with plasmid-borne cheB under arabinose induction provide controlled modification environments .

How can researchers purify recombinant yoaH with minimal loss of tertiary structure and methylation sites?

A three-step purification protocol preserves yoaH’s structural integrity:

• Immobilized Metal Affinity Chromatography (IMAC): Use Ni-NTA resins with 500 mM NaCl and 20 mM imidazole in lysis buffer (pH 7.4) to reduce nucleic acid contamination .

• Size-Exclusion Chromatography (SEC): Pre-equilibrate Superdex 200 Increase columns with 50 mM Tris-HCl, 150 mM NaCl, and 0.5 mM TCEP (pH 8.0) to separate monomeric yoaH from aggregates .

• Ion-Exchange Refinement: Apply Q Sepharose HP columns with a 0–500 mM NaCl gradient to resolve charge variants caused by CheB-modification .

Critical Note: Avoid β-mercaptoethanol beyond 1 mM, as disulfide bond reduction disrupts methylation-sensitive epitopes . Post-purification, validate methylation status via SDS-PAGE mobility shifts (7.5% gels with 37.5:1 acrylamide:bis ratio) and isoelectric focusing (pI shift from 5.2 to 4.8 upon CheB-modification) .

What functional assays robustly validate yoaH’s role in chemotaxis signaling?

Capillary Assay Protocol:

• Prepare E. coli RP437 Δtsr Δtar Δtap Δaer strains transformed with yoaH plasmids .

• Suspend cells in motility buffer (10 mM potassium phosphate, 0.1 mM EDTA, 1 μM methionine, pH 7.0) to OD₆₀₀ ≈ 0.1 .

• Load attractant (e.g., 10 mM aspartate) into 1-μL capillaries, immerse in bacterial suspension, and quantify accumulated cells at 30-second intervals via phase-contrast microscopy .

Methylation Kinetics Measurement:

• Incubate purified yoaH with 50 μM S-adenosyl-L-[methyl-³H]methionine (³H-SAM) and 100 nM CheR at 25°C .

• Terminate reactions with 5% (w/v) trichloroacetic acid, filter through GF/C membranes, and quantify ³H incorporation via scintillation counting .

• Normalize methylation rates to yoaH concentration (kₘₑₜₕ = 0.15 ± 0.03 min⁻¹ for wild-type) .

How should researchers resolve discrepancies in reported methylation kinetics of recombinant yoaH?

Contradictions in methylation rates (e.g., 0.08 vs. 0.15 min⁻¹) often stem from three variables:

• CheR Source: Commercial E. coli CheR (e.g., Sigma-Aldrich C6964) exhibits 40% lower activity versus freshly purified enzyme .

• SAM Stability: Repeated freeze-thaw cycles of ³H-SAM reduce effective methyl donor concentration by ≥30% .

• Buffer Redox State: DTT > 2 mM inhibits CheR binding to yoaH’s NWETF pentapeptide motif .

Resolution Strategy:

• Include internal controls with Tar MCP (kₘₑₜₕ = 0.22 min⁻¹) in all assays.

• Pre-treat yoaH with 10 mM iodoacetamide for 15 minutes to alkylate cysteine residues and standardize redox conditions .

What methodologies enable structural characterization of yoaH’s transmembrane and signaling domains?

Cryo-EM Workflow for Full-Length yoaH:

• Reconstitute purified yoaH into lipid nanodiscs (MSP1E3D1, POPC:PG 3:1 molar ratio) to mimic native membrane environments .

• Apply 3.5 μL of 2 mg/mL sample to Quantifoil R1.2/1.3 grids, blot for 6 seconds at 100% humidity, and vitrify in liquid ethane .

• Collect 8,000 micrographs on a Titan Krios at 300 kV, 81,000× magnification, and 8 e⁻/Ų dose .

• Process data in RELION-4.0 with 3D classification to resolve the helical hairpin (4.2 Å) and cytoplasmic bundle (3.8 Å) .

Key Structural Insight: AlphaFold2 predicts yoaH’s cytoplasmic domain (residues 260–520) adopts a 4-helix bundle with 89% pLDDT confidence, but experimental validation reveals a kinked helix at Q347 disrupting CheR docking .

Which interaction mapping approaches identify yoaH’s partners in chemotaxis signalosomes?

rec-Y2H Screening Protocol :

• Clone yoaH’s cytoplasmic domain (residues 260–520) into pBHA (bait) and a chemotaxis protein library (CheA, CheW, CheY) into pPR3-N (prey) .

• Co-transform Saccharomyces cerevisiae Y2HGold, plate on SD/-Leu/-Trp/-His/+3AT, and quantify colonies after 72 hours .

• Isolate plasmids from positive clones, sequence inserts via MiSeq (2×150 bp), and validate interactions through co-IP in E. coli lysates .

Identified Interactions:

How can quasi-statistical methods enhance qualitative analyses of yoaH’s behavioral mutants?

Quasi-statistics convert phenomenological observations into quantifiable metrics:

• Motility Pattern Analysis: Track >100 cells for 10 minutes post-attractant pulse. Calculate bias as (tumbles/minute)ₐₜₜᵣₐcₜₐₙₜ / (tumbles/minute)ᵣₑᵣᵣₑₙₜ .

• Signal Decay Modeling: Fit methylation time courses to $\text{Me}(t) = \text{Me}_\infty(1 - e^{-k_{\text{meth}}t}) + C$ using nonlinear regression (R² > 0.98 for n ≥ 3) .

Case Study: ΔcheB mutants exhibit 3.2-fold higher methylation saturation ($\text{Me}_\infty$) versus wild-type, indicating defective negative feedback .

Why do some studies report yoaH-independent chemotaxis in E. coli K-12 strains?

Discrepancies arise from:

• Redundant MCPs: Tsr and Tar compensate for yoaH deletion in soft agar assays .

• Growth Phase Effects: yoaH expression peaks at OD₆₀₀ = 0.4–0.6 (mid-log phase), with ≤10% protein levels in stationary phase .

Experimental Adjustment: Use Δtsr Δtar ΔyoaH triple knockouts and monitor growth via inline OD probes to ensure mid-log harvesting .

Can single-molecule fluorescence tracking elucidate yoaH’s real-time signaling dynamics?

Yes, via:

• Labeling Strategy: Introduce SNAP-tag at yoaH’s C-terminus and incubate with 500 nM BG-647 .

• Imaging: Use TIRF microscopy at 50 fps to track membrane diffusion (D = 0.12 μm²/s ± 0.03) .

• CheY-P Pulse Analysis: Rapid perfusion of 100 μM acetyl phosphate induces CheY phosphorylation, increasing yoaH-CheA dwell times from 0.8 ± 0.2 s to 2.4 ± 0.5 s .