Recombinant Acinetobacter baumannii UPF0060 membrane protein A1S_1909 (A1S_1909)

What is the amino acid sequence of A1S_1909 and what does it reveal about protein structure?

The full amino acid sequence of A1S_1909 is: MFGLFIITAIAEILGCYFPYLILKEGKSAWLWLPTALSLAVFVWLLTLHPAASGRIYAAYGGIYIFTALMWLRFVDQVALTRWDILGGVIVLCGAGLIILQPQGLIR . Hydropathy analysis of this sequence reveals multiple hydrophobic regions characteristic of transmembrane domains, suggesting A1S_1909 is an integral membrane protein. The presence of glycine-rich motifs (particularly in positions 84-98) may indicate flexible regions important for membrane insertion or protein-protein interactions.

To analyze the structural characteristics:

• Conduct hydrophobicity plotting using Kyte-Doolittle or similar algorithms

• Perform secondary structure prediction (PSIPRED, JPred) to identify potential alpha-helical transmembrane segments

• Use homology modeling against known UPF0060 family members to generate structural hypotheses

What expression systems are most effective for recombinant A1S_1909 production?

While A1S_1909 is natively expressed in Acinetobacter baumannii, recombinant expression typically employs E. coli systems due to their high yield and ease of genetic manipulation . The most effective experimental approach involves:

• Expression vector selection: pET vectors with T7 promoter systems provide high-level, inducible expression

• Host strain optimization: BL21(DE3) derivatives, particularly C41(DE3) or C43(DE3), are designed for membrane protein expression

• Fusion tag implementation: N-terminal His-tags facilitate purification while minimizing interference with membrane insertion

For challenging membrane proteins like A1S_1909, consider the following experimental design parameters:

What are the critical quality control parameters for verifying recombinant A1S_1909 identity and purity?

Quality assessment of recombinant A1S_1909 requires multiple complementary approaches :

• Purity assessment: SDS-PAGE analysis should confirm >90% purity with the expected molecular weight (~12 kDa plus tag size)

• Identity confirmation: Western blotting using anti-His antibodies or mass spectrometry analysis of tryptic digests

• Structural integrity: Circular dichroism spectroscopy to verify secondary structure content (expected high alpha-helical content)

• Functional validation: Reconstitution into proteoliposomes to assess membrane insertion capability

Each batch should be documented with:

• Concentration determination using both Bradford/BCA assays and A280 measurements

• Endotoxin testing if intended for immunological studies

• Freeze-thaw stability assessment

What are the optimal storage conditions for maintaining A1S_1909 stability?

A1S_1909 requires specific storage conditions to maintain structural integrity and function :

• Long-term storage: Store lyophilized protein at -20°C to -80°C in airtight containers to prevent moisture absorption

• Working aliquots: Store reconstituted protein at 4°C for up to one week to minimize freeze-thaw cycles

• Buffer composition: Tris/PBS-based buffer supplemented with 6% trehalose at pH 8.0 provides optimal stability

For extended storage periods, implement a cryoprotectant strategy:

• Add glycerol to a final concentration of 5-50% (with 50% being optimal for most applications)

• Divide into single-use aliquots (typically 50-100 μL) to prevent repeated freeze-thaw cycles

• Document stability through activity assays at regular time intervals

What reconstitution protocol maximizes A1S_1909 functionality?

Proper reconstitution is critical for membrane protein functionality :

• Centrifuge the vial briefly before opening to collect all lyophilized material

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

• Allow complete dissolution by gentle rotation (not vortexing) for 10-15 minutes at room temperature

• For applications requiring higher concentrations, reconstitute initially at 1 mg/mL and concentrate using appropriate molecular weight cutoff concentrators

For membrane integration studies, consider:

• Direct reconstitution into detergent micelles (0.1% DDM or LDAO)

• Stepwise dialysis into lipid vesicles composed of E. coli polar lipid extracts

• Validation of proper folding using intrinsic tryptophan fluorescence

How should researchers design experiments to investigate A1S_1909's membrane topology?

Investigating membrane topology requires a multi-technique approach :

• Proteolytic accessibility mapping:

  • Reconstitute A1S_1909 into proteoliposomes

  • Treat with membrane-impermeable proteases (e.g., trypsin)

  • Analyze protected fragments by mass spectrometry to identify membrane-embedded regions

• Cysteine scanning mutagenesis:

  • Generate single-cysteine variants throughout the protein sequence

  • Label with membrane-permeable and impermeable thiol-reactive probes

  • Compare labeling patterns to map membrane-accessible regions

• Computational prediction validation:

  • Generate topology models using algorithms (TMHMM, TOPCONS)

  • Design experiments to test specific loop regions

  • Correlate findings with evolutionary conservation patterns

What approaches can determine if A1S_1909 forms oligomeric structures?

Membrane protein oligomerization analysis requires specialized techniques :

• Chemical crosslinking:

  • Treat purified A1S_1909 with membrane-compatible crosslinkers (DSS, BS3)

  • Analyze crosslinked products by SDS-PAGE and mass spectrometry

  • Identify interaction interfaces through crosslink mapping

• Analytical ultracentrifugation:

  • Analyze sedimentation equilibrium patterns in detergent solutions

  • Calculate molecular weights of protein-detergent complexes

  • Determine stoichiometry of oligomeric assemblies

• FRET analysis:

  • Generate fluorescently labeled A1S_1909 variants

  • Measure energy transfer efficiencies between labels

  • Reconstruct three-dimensional arrangements of subunits

The experimental design should incorporate appropriate controls as outlined in standard protocols :

• Negative controls: non-interacting membrane proteins of similar size

• Positive controls: known oligomeric membrane proteins

• Concentration dependence: test across physiologically relevant concentrations

How can researchers assess A1S_1909 interactions with other bacterial proteins?

Protein-protein interaction studies for membrane proteins require specialized approaches :

• Bacterial two-hybrid systems:

  • Adapt membrane-specific two-hybrid methods (BACTH)

  • Screen against genomic libraries from A. baumannii

  • Validate interactions using co-immunoprecipitation

• Proximity labeling approaches:

  • Generate A1S_1909 fusions with BioID or APEX2 enzymes

  • Express in native bacterial environment

  • Identify proximal proteins through biotinylation and mass spectrometry

• Co-purification strategies:

  • Perform tandem affinity purification with tagged A1S_1909

  • Analyze co-purifying proteins by mass spectrometry

  • Confirm specific interactions through reciprocal tagging

Experimental design considerations include :

• Detergent selection to maintain native interactions

• Controls for non-specific binding

• Validation in multiple experimental systems

What experimental approaches can determine A1S_1909's role in bacterial membrane physiology?

Investigating physiological functions requires complementary genetic and biochemical approaches:

• Gene knockout/knockdown studies:

  • Generate A1S_1909 deletion mutants in A. baumannii

  • Perform comprehensive phenotypic profiling:

    • Growth rates in various media

    • Membrane permeability assays

    • Resistance to environmental stressors

• Complementation analysis:

  • Reintroduce wild-type or mutant A1S_1909 into knockout strains

  • Assess restoration of phenotypes

  • Correlate structure-function relationships

• Metabolomic profiling:

  • Compare metabolite profiles between wild-type and A1S_1909 mutants

  • Identify metabolic pathways affected

  • Generate hypotheses about transport or signaling functions

The experimental design should incorporate :

• Multiple independent mutant lines

• Growth under various stress conditions

• Appropriate statistical analysis of phenotypic data

How can researchers investigate A1S_1909's potential role in antimicrobial resistance?

A systematic approach to investigating antimicrobial resistance connections includes:

• Susceptibility testing:

  • Compare minimum inhibitory concentrations between wild-type and A1S_1909 mutants

  • Test against diverse antibiotic classes

  • Analyze time-kill kinetics for mechanistic insights

• Membrane permeability assessment:

  • Measure uptake of fluorescent probes (e.g., NPN, SYTOX Green)

  • Analyze membrane potential using voltage-sensitive dyes

  • Correlate changes with antibiotic accumulation

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type and mutants

  • Focus on known resistance determinants

  • Identify regulatory networks potentially involving A1S_1909

Data analysis should include :

• Time-course experiments to capture adaptation processes

• Dose-response relationships

• Integration of multiple data types for comprehensive models

How should researchers design site-directed mutagenesis experiments to probe A1S_1909 structure-function relationships?

Strategic mutagenesis requires careful experimental design :

• Targeting strategy:

  • Conserved residues within the UPF0060 family

  • Predicted functional sites (e.g., charged residues in transmembrane regions)

  • Known motifs for membrane protein folding/assembly

• Mutation types:

  • Conservative substitutions to probe specific interactions

  • Charge inversions to disrupt electrostatic interactions

  • Alanine scanning of predicted functional loops

• Functional assessment:

  • Expression and folding analysis

  • Membrane integration efficiency

  • Phenotypic complementation in knockout strains

What bioinformatic approaches help understand A1S_1909's evolutionary relationships and functional predictions?

Comprehensive evolutionary analysis includes :

• Phylogenetic profiling:

  • Identify A1S_1909 homologs across bacterial species

  • Correlate presence/absence with specific phenotypes

  • Analyze gene neighborhood conservation

• Coevolution analysis:

  • Perform multiple sequence alignment of UPF0060 family members

  • Identify coevolving residue pairs using methods like GREMLIN or EVcouplings

  • Predict structural contacts and functional interactions

• Structural threading and modeling:

  • Generate structural models using resources like I-TASSER or AlphaFold

  • Validate predictions with experimental constraints

  • Identify potential binding sites or functional domains

Analytical considerations include:

• Appropriate sequence similarity thresholds

• Consideration of horizontal gene transfer events

• Integration with experimental validation strategies

What are the challenges and solutions in crystallizing or obtaining structural data for A1S_1909?

Membrane protein structural biology presents specific challenges:

• Crystallization strategies:

  • Screening stabilizing detergents (DDM, LDAO, GDN)

  • Exploring lipidic cubic phase methods

  • Testing fusion partners (T4 lysozyme, BRIL) to enhance crystal contacts

• Cryo-EM approaches:

  • Reconstitution into nanodiscs or amphipols

  • Optimizing particle size through oligomerization

  • Implementing focused refinement for flexible regions

• NMR-based methods:

  • Uniform or selective isotope labeling strategies

  • Detergent micelle optimization for solution NMR

  • Solid-state NMR approaches for reconstituted samples