Recombinant Yersinia pestis bv. Antiqua UPF0266 membrane protein YPA_1127 (YPA_1127)

What is YPA_1127 and what are its basic properties?

YPA_1127 is a UPF0266 family membrane protein found in Yersinia pestis biovar Antiqua. It is a full-length protein consisting of 153 amino acids with the sequence: "MSVTDLVLVVFIALLLIYAIYDEFIMNMMKGKTRLQVHLKRKNKLDCMIFVGLIGILIYNVMAHGAPLTTYLLVGLALVAVYISYIRWPKLLFKNTGFFYANTFIEYSRIKSMNLSEDGILVIDLEQRRLLIQVKKLDDLEKIYNFFIENQS" . The protein is identified by UniProt ID Q1C8X8 and is characterized as a membrane-associated protein, suggesting its involvement in membrane-related processes . Structural analysis indicates multiple hydrophobic regions consistent with transmembrane domains, which is typical of membrane proteins.

How should researchers approach initial characterization of YPA_1127?

Initial characterization should begin with bioinformatic analysis to predict protein domains, potential post-translational modifications, and transmembrane regions. For experimental characterization, researchers should:

• Perform SDS-PAGE analysis to confirm the protein's molecular weight and purity (>90% purity is recommended for most applications)

• Conduct Western blotting using anti-His tag antibodies to verify expression

• Employ circular dichroism (CD) to assess secondary structure composition

• Use dynamic light scattering to evaluate protein homogeneity

When working with membrane proteins like YPA_1127, it's crucial to maintain appropriate detergent concentrations throughout the analysis process to prevent protein aggregation. Experimental design should include appropriate controls to account for the effects of detergents on analytical techniques .

What are the optimal conditions for expressing recombinant YPA_1127?

The recombinant YPA_1127 protein is typically expressed in E. coli with an N-terminal His tag . For optimal expression, consider the following methodology:

• Select an appropriate E. coli strain (BL21(DE3) is commonly used for membrane proteins)

• Use a vector with a strong promoter (T7 or tac) and codon optimization for E. coli

• Culture conditions: LB media supplemented with appropriate antibiotics

• Induction parameters: 0.5-1.0 mM IPTG at OD600 of 0.6-0.8

• Post-induction cultivation: 16-18°C for 16-20 hours to allow proper folding

Membrane proteins often present expression challenges due to hydrophobicity and potential toxicity to host cells . Low-temperature induction and the addition of membrane-stabilizing compounds (such as 5% glycerol) to the culture medium can improve expression yields. Expression levels should be monitored via small-scale test expressions before scaling up.

What advanced purification techniques are most effective for obtaining high-purity YPA_1127?

For advanced purification of YPA_1127, implement a multi-step chromatography approach:

• Initial capture: Ni-NTA affinity chromatography using the N-terminal His tag

  • Use gradual imidazole concentration increases (20-250 mM) to separate full-length protein from truncated forms

  • Include 0.05-0.1% appropriate detergent (DDM or LDAO) in all buffers

• Intermediate purification: Size exclusion chromatography

  • Use Superdex 200 column equilibrated with buffer containing 0.05% detergent

  • Monitor peak fractions for protein purity and oligomeric state

• Polishing: Ion exchange chromatography (if necessary)

  • Select appropriate resin based on theoretical pI of YPA_1127

For advanced applications requiring exceptionally pure protein, consider implementing additional techniques such as hydroxyapatite chromatography or affinity tag removal followed by reverse affinity purification. The purity should be >90% as determined by SDS-PAGE for most applications .

What methods are appropriate for analyzing the membrane topology of YPA_1127?

To analyze membrane topology of YPA_1127, employ a combination of computational prediction and experimental validation:

• Computational prediction:

  • Use membrane protein topology prediction algorithms (TMHMM, Phobius, TOPCONS)

  • Apply hydropathy plot analysis (Kyte-Doolittle) to identify transmembrane segments

• Experimental validation:

  • Cysteine scanning mutagenesis with membrane-impermeable labeling reagents

  • Protease protection assays with reconstituted proteoliposomes

  • Site-directed fluorescence labeling combined with quenching studies

• Advanced structural techniques:

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

  • Electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling

When designing experiments, consider using a data.table structure in R to efficiently organize and analyze topology mapping data . This approach allows for rapid comparison between computational predictions and experimental results.

How can researchers investigate potential binding partners or substrates of YPA_1127?

To identify binding partners or substrates, employ the following methodological approaches:

• Pull-down assays:

  • Use His-tagged YPA_1127 immobilized on Ni-NTA resin

  • Incubate with Y. pestis lysate or subcellular fractions

  • Analyze co-precipitating proteins by mass spectrometry

• Crosslinking studies:

  • Apply membrane-permeable crosslinkers of various spacer lengths

  • Identify crosslinked complexes via Western blotting and mass spectrometry

• Lipidomic analysis:

  • Evaluate lipid binding preferences using liposome flotation assays

  • Perform thin-layer chromatography with bound lipids extracted from purified protein

• Functional reconstitution:

  • Incorporate YPA_1127 into proteoliposomes with potential substrates

  • Monitor transport or enzymatic activity under various conditions

For data analysis, implement appropriate statistical methods to distinguish specific interactions from background. Consider using membrane extracts from Y. pestis cultured under different conditions to identify condition-specific interactions.

What control experiments are essential when studying YPA_1127 function?

When designing experiments to study YPA_1127 function, include these essential controls:

• Negative controls:

  • Empty vector-transformed E. coli processed identically to YPA_1127-expressing cells

  • Irrelevant membrane protein of similar size with the same tag

  • Heat-denatured YPA_1127 to control for non-specific effects

• Positive controls:

  • Well-characterized membrane protein from the same family

  • Native YPA_1127 isolated from Y. pestis (if feasible)

• Technical controls:

  • Detergent-only samples to account for detergent effects

  • Tag-only protein to distinguish tag-related artifacts

A robust experimental design requires significant planning to ensure control over the testing environment, sound experimental treatments, and proper assignment of subjects to treatment groups . Without proper planning, unexpected external variables can alter experimental outcomes.

How should researchers design experiments to investigate the role of YPA_1127 in bacterial pathogenesis?

To investigate YPA_1127's role in pathogenesis, implement a multi-faceted experimental design:

• Gene knockout/knockdown approaches:

  • Generate YPA_1127 deletion mutants in Y. pestis

  • Create complemented strains expressing wild-type or mutant YPA_1127

  • Perform in vitro and in vivo virulence assays comparing wild-type, mutant, and complemented strains

• Protein-protein interaction studies:

  • Apply bacterial two-hybrid systems

  • Conduct co-immunoprecipitation with host cell lysates

  • Perform proximity labeling in infected cells

• Host response analysis:

  • Compare host cell transcriptomes upon infection with wild-type vs. YPA_1127 mutant

  • Evaluate changes in host cell membrane properties and signaling pathways

• Structure-function relationship studies:

  • Generate point mutations in conserved residues

  • Create chimeric proteins with homologs from less virulent species

Follow the principles of true experimental design by including randomization, proper controls, and blinding where appropriate . The experimental design should provide unbiased estimates of inputs and enable the detection of differences caused by independent variables.

How can advanced membrane protein reconstitution methods be applied to study YPA_1127?

For advanced functional characterization, reconstitute YPA_1127 using these methodologies:

• Nanodiscs preparation:

  • Select appropriate membrane scaffold proteins (MSPs)

  • Optimize lipid composition based on Y. pestis membrane composition

  • Use a gradual detergent removal approach via dialysis or adsorption

• Proteoliposome reconstitution:

  • Optimize protein-to-lipid ratios (typically 1:100 to 1:1000 by weight)

  • Control liposome size using extrusion through defined pore-size membranes

  • Verify protein orientation using protease protection assays

• Advanced reconstitution systems:

  • Polymer-supported bilayers for surface-sensitive techniques

  • Droplet interface bilayers for electrical measurements

  • Microfluidic systems for high-throughput functional assays

These reconstitution methods provide controlled environments for studying membrane protein function while maintaining native-like lipid surroundings. For optimal results, characterized reconstituted systems using multiple techniques (electron microscopy, dynamic light scattering, fluorescence microscopy) to ensure homogeneity and proper protein incorporation.

What are the latest techniques for investigating conformational changes in membrane proteins like YPA_1127?

To study conformational dynamics of YPA_1127, consider these advanced biophysical approaches:

• Single-molecule FRET:

  • Introduce fluorophore pairs at strategic positions

  • Monitor distance changes under various conditions

  • Analyze conformational populations and transition kinetics

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

  • Compare deuterium uptake patterns under different conditions

  • Identify regions undergoing conformational changes

  • Develop mathematical models of protein dynamics using HDX data

• Molecular dynamics simulations:

  • Build atomistic models based on homology or predicted structures

  • Simulate protein behavior in membrane environments

  • Identify potential conformational states and transition pathways

• Cryo-electron microscopy:

  • Capture YPA_1127 in different functional states

  • Determine high-resolution structures of each state

  • Map conformational changes to functional mechanisms

When implementing these techniques, use appropriate data analysis frameworks like data.table in R to manage complex datasets and extract meaningful patterns . Integrate results from multiple approaches to build comprehensive models of YPA_1127 conformational dynamics.

What are the optimal conditions for long-term storage of purified YPA_1127?

For optimal storage of purified YPA_1127, follow these methodological guidelines:

• Short-term storage (1-2 weeks):

  • Store at 4°C in purification buffer containing 0.02-0.05% detergent

  • Add protease inhibitors to prevent degradation

  • Maintain protein at concentrations below aggregation threshold (typically 1-5 mg/mL)

• Long-term storage:

  • Store at -20°C/-80°C in small aliquots to avoid freeze-thaw cycles

  • Add 5-50% glycerol as a cryoprotectant (50% glycerol is recommended)

  • Lyophilization in buffer containing stabilizing agents and disaccharides

• Quality monitoring:

  • Perform regular SDS-PAGE analysis to check for degradation

  • Use dynamic light scattering to monitor aggregation state

  • Verify activity using appropriate functional assays

Repeated freeze-thaw cycles should be avoided as they can lead to protein aggregation and loss of function . For aliquots in active use, store at 4°C for up to one week.

What quality control measures should be implemented for YPA_1127 preparations?

To ensure consistent quality of YPA_1127 preparations, implement these quality control procedures:

• Purity assessment:

  • SDS-PAGE analysis (target: >90% purity)

  • Size exclusion chromatography to evaluate monodispersity

  • Mass spectrometry to confirm protein identity and detect modifications

• Functional validation:

  • Binding assays with known ligands or interacting partners

  • Activity assays based on predicted function

  • Circular dichroism to confirm proper secondary structure

• Stability testing:

  • Thermal shift assays to determine melting temperature

  • Time-course stability at various temperatures

  • Detergent screening to identify optimal stabilizing conditions

• Batch consistency:

  • Establish reference standards for each quality parameter

  • Implement statistical process control to monitor batch-to-batch variation

  • Document preparation conditions thoroughly for reproducibility

Maintain detailed records of each preparation using standardized protocols and quality metrics. This approach ensures that experimental results remain comparable across different studies and research groups.