Recombinant Acinetobacter sp. Aliphatic sulfonates import ATP-binding protein SsuB (ssuB)

What is the basic structure and function of the SsuB protein in Acinetobacter species?

SsuB belongs to the ATP-binding cassette (ABC) family, a group of proteins that actively transport various substrates across cellular membranes. As the ATP-binding component of the aliphatic sulfonates import system, SsuB provides the energy required for substrate translocation through ATP hydrolysis. Similar to other ABC transporters, SsuB likely contains conserved motifs including Walker A and B sequences, signature C motifs, and D, H, and Q loops that are critical for nucleotide binding and hydrolysis. The protein functions in conjunction with transmembrane domains to facilitate the import of aliphatic sulfonates, which can serve as alternative sulfur sources for the organism under sulfate-limiting conditions .

What expression systems are most suitable for recombinant SsuB production in laboratory settings?

Based on successful approaches with other Acinetobacter proteins, the pET expression system using E. coli BL21(DE3) as a host is often the first choice for initial expression trials. This system offers tight regulation through the T7lac inducible promoter with IPTG induction. For recombinant SsuB expression, researchers should consider several factors:

• Vector selection: pET-22b(+) with appropriate affinity tags (His-tag) facilitates purification

• Expression host: E. coli BL21(DE3) strains are typically used for their reduced protease activity

• Induction conditions: Optimized IPTG concentration (typically 0.1-1 mM) and temperature (often 16-25°C for membrane-associated proteins)

• Growth media: Rich media such as LB or TB for high cell density

The expression construct should be verified via colony PCR and nucleotide sequencing before transformation into the expression host .

How can the accessibility of translation initiation sites affect SsuB expression levels?

Translation initiation site accessibility significantly impacts recombinant protein expression success. Research analyzing 11,430 expression experiments shows that the accessibility of translation initiation sites (modeled using mRNA base-unpairing across Boltzmann's ensemble) strongly predicts expression outcomes. For SsuB protein, optimizing the accessibility of the start codon and surrounding nucleotides can dramatically improve translation efficiency.

The opening energy (ΔGopen) of the translation initiation region correlates with expression success, with lower energy barriers leading to higher expression levels. Synonymous substitutions in the first 9 codons can be strategically implemented to modulate this accessibility without altering the amino acid sequence. Tools like TIsigner can help design optimized sequences with improved accessibility profiles, potentially increasing SsuB production yields by 3-4 fold in some cases .

What true experimental design approaches are most effective for optimizing recombinant SsuB production parameters?

A robust true experimental design for optimizing SsuB production should incorporate random assignment, control groups, and manipulation of independent variables. Consider implementing:

• Factorial designs to simultaneously evaluate multiple factors affecting SsuB expression:

  • Temperature (16°C, 25°C, 37°C)

  • Induction timing (early, mid, late log phase)

  • Inducer concentration (0.1, 0.5, 1.0 mM IPTG)

  • Media composition (minimal vs. rich media)

• Response surface methodology to identify optimal conditions and potential interactions between variables

• Control groups including:

  • Empty vector controls

  • Expression of known high-yielding proteins (positive control)

  • Non-induced cultures

This approach ensures internal validity through random assignment of culture conditions and controlled manipulation of variables. Blocking designs can help control for extraneous factors like batch effects or equipment variations. Measurement of dependent variables should include both protein yield and activity assessments to ensure functional protein production .

How can researchers assess and improve the solubility of recombinant SsuB protein?

SsuB, as an ATP-binding protein, may present solubility challenges during recombinant expression. A comprehensive approach to improving solubility includes:

Assessment methods:

• Small-scale expression trials with fractionation analysis (soluble vs. insoluble)

• SDS-PAGE and Western blot analysis of supernatant and pellet fractions

• Activity assays to confirm functional folding (ATP binding/hydrolysis)

Solubility enhancement strategies:

• Expression temperature optimization (16-25°C often improves folding)

• Co-expression with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Fusion tags (MBP, SUMO, Thioredoxin) to enhance solubility

• Buffer optimization during lysis and purification:

  Buffer Component	Range to Test	Purpose
  NaCl	100-500 mM	Ionic strength
  Glycerol	5-20%	Stabilization
  Detergents	0.1-1%	Membrane protein solubilization
  Reducing agents	1-10 mM DTT/BME	Prevent oxidation

• Additives screening for stabilization (ATP, Mg²⁺, specific substrates)

What are the critical considerations for designing a purification strategy for recombinant SsuB?

Purifying functional SsuB requires a carefully designed strategy that preserves the protein's native conformation and activity. Consider these critical factors:

• Affinity tag selection: C-terminal His-tags are commonly used with pET vectors, minimizing interference with the N-terminal nucleotide-binding domain common in ABC transporters

• Cell lysis conditions: Gentle lysis using enzymatic methods (lysozyme) or moderate sonication to prevent protein aggregation

• Multi-step purification approach:

  • Immobilized metal affinity chromatography (IMAC) as initial capture step

  • Ion exchange chromatography for intermediate purification

  • Size exclusion chromatography as polishing step

• Buffer optimization throughout the purification process:

  • Inclusion of ATP/ADP (0.1-1 mM) for stabilization

  • Magnesium ions (1-5 mM) as cofactors

  • Appropriate pH range (typically 7.0-8.0 for ABC proteins)

• Quality control assessments:

  • Purity evaluation via SDS-PAGE and Western blot

  • Functional assessment through ATP binding/hydrolysis assays

  • Thermal stability analysis to guide buffer optimization

Monitor protein activity throughout purification to ensure the isolated protein retains its functional properties .

What techniques are most appropriate for analyzing ATP binding and hydrolysis activity of purified SsuB?

As an ATP-binding protein, SsuB's primary function involves nucleotide binding and hydrolysis. Several complementary techniques can assess these activities:

ATP Binding Assays:

• Fluorescence-based methods:

  • Intrinsic tryptophan fluorescence quenching upon nucleotide binding

  • TNP-ATP binding (fluorescent ATP analog) with increased emission upon protein binding

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of binding thermodynamics

  • Determination of dissociation constants (Kd), stoichiometry, and enthalpy changes

ATP Hydrolysis Assays:

• Inorganic phosphate (Pi) release measurements:

  • Malachite green colorimetric assay

  • EnzChek phosphate assay (more sensitive)

• Coupled enzyme assays:

  • Pyruvate kinase/lactate dehydrogenase system linking ATP hydrolysis to NADH oxidation

  • Continuous spectrophotometric monitoring at 340 nm

Experimental Design Considerations:

• Temperature control (typically 25°C or 37°C)

• Inclusion of appropriate divalent cations (Mg²⁺, Mn²⁺)

• Testing ATP concentration ranges (10 μM to 5 mM)

• Kinetic parameter determination (Km, Vmax, kcat)

These assays should be performed under conditions that mimic the protein's physiological environment, including appropriate pH, salt concentration, and presence of potential allosteric regulators .

How can researchers investigate the interaction between SsuB and membrane components of the transport system?

SsuB's function depends on its interaction with membrane components to form a complete transport complex. Investigating these interactions requires specialized approaches:

Reconstitution Systems:

• Proteoliposome reconstitution with purified membrane components

• Nanodiscs to isolate and study specific protein-protein interactions

• Detergent-solubilized complexes for initial characterization

Interaction Analysis Techniques:

• Pull-down assays using affinity-tagged components

• Surface plasmon resonance (SPR) for real-time interaction kinetics

• Crosslinking studies to capture transient interactions

• Förster resonance energy transfer (FRET) for dynamic studies in native-like environments

Functional Reconstitution:

• Transport assays using fluorescent or radiolabeled substrate analogs

• Assessment of ATP hydrolysis in the presence of transport substrates and membrane components

• Evaluation of coupling efficiency between ATP hydrolysis and substrate transport

These studies should be complemented with structural analyses when possible, including negative-stain electron microscopy or cryo-EM to visualize the assembled complex .

How should researchers address expression failures of recombinant SsuB protein?

Expression failures are common challenges in recombinant protein production, with approximately 50% of recombinant proteins failing to express in various host cells. When encountering difficulties with SsuB expression, implement this systematic troubleshooting approach:

• Translation initiation optimization:

  • Analyze mRNA secondary structure around the start codon using computational tools

  • Implement synonymous codon changes in the first 9 codons to improve accessibility

  • Consider using TIsigner or similar tools to design optimized sequences with improved accessibility profiles

• Expression strain evaluation:

  • Test multiple E. coli strains (BL21, C41/C43, Rosetta for rare codons)

  • Consider specialized strains for membrane-associated proteins

  • Evaluate different growth media formulations (TB, SOB, M9 minimal)

• Induction parameter adjustment:

  • Reduce induction temperature (16-25°C)

  • Lower inducer concentration (0.01-0.1 mM IPTG)

  • Test auto-induction media to provide gradual induction

• Vector and construct modifications:

  • Try alternative promoter systems (tac, arabinose)

  • Test different fusion tags (MBP, GST, SUMO)

  • Consider codon optimization for the entire gene sequence

Remember that apparently negative results in initial SDS-PAGE analysis might still represent successful but low-level expression that can be detected by more sensitive methods like Western blotting or activity assays .

What bioinformatic approaches can improve success rates in recombinant SsuB expression and characterization?

Bioinformatic analyses provide valuable insights that can guide experimental design and troubleshooting for SsuB expression:

• Sequence-based predictions:

  • Transmembrane domain prediction to identify potential membrane-associated regions

  • Secondary structure analysis to identify structured domains

  • Identification of conserved ABC transporter motifs (Walker A/B, signature C, D/H/Q loops)

• Expression optimization tools:

  • Translation initiation site accessibility analysis

  • Codon usage optimization for expression host

  • mRNA stability prediction and optimization

  • Identification and removal of internal Shine-Dalgarno-like sequences

• Comparative analysis:

  • Structure-guided alignments with characterized ABC transporters

  • Homology modeling based on crystal structures of related proteins

  • Identification of potential regulatory elements or post-translational modifications

• Experimental design guidance:

  • Domain boundary prediction for construct design

  • Identification of stable subdomains for expression

  • Structure-based rational mutation design for functional studies

Applied systematically, these approaches can increase success rates from ~50% to >80% for challenging proteins like SsuB .

How can researchers evaluate the impact of SsuB expression on host cell physiology and optimize production strategies accordingly?

Recombinant protein overexpression creates metabolic burden that can significantly impact host cell physiology and ultimately affect protein yield and quality. Understanding and managing these effects is crucial for optimizing SsuB production:

• Physiological monitoring:

  • Growth curve analysis comparing induced vs. non-induced cultures

  • Measurement of final cell density (OD600)

  • Viability assessment using flow cytometry or plate counts

  • Monitoring of stress response markers (e.g., heat shock proteins)

• Metabolic burden assessment:

  • Analysis of cellular growth rates post-induction

  • Quantification of biomass yield coefficients

  • Measurement of central carbon metabolism fluxes

  • Evaluation of ribosomal capacity utilization

• Optimization strategies based on physiological data:

  • Implementation of fed-batch cultivation to control growth rates

  • Design of induced-growth rate control strategies

  • Strategic timing of induction based on growth phase

  • Temperature shifting protocols to balance growth and expression

• Stochastic simulation modeling:

  • Prediction of protein production rates and cell growth constraints

  • Evaluation of trade-offs between protein yield and host metabolism

  • Testing hypotheses about resource allocation during overexpression

Research indicates that higher accessibility of translation initiation sites leads to higher protein production but slower cell growth, supporting the concept of protein cost where cell growth is constrained by protein circuits during overexpression. Balancing these factors is key to optimal production strategies .

What structural features of SsuB are critical for its ATP-binding and hydrolysis functions?

As an ATP-binding protein of the ABC transporter family, SsuB contains several highly conserved structural elements that are essential for its function:

• Core nucleotide-binding domain architecture:

  • RecA-like subdomain containing Walker A and B motifs

  • Helical subdomain containing the signature C-motif (LSGGQ)

  • These domains form a "sandwich dimer" arrangement in functional state

• Conserved sequence motifs and their functions:

  Motif	Consensus Sequence	Function
  Walker A	GXXGXGKS/T	Phosphate binding
  Walker B	ɸɸɸɸDE (ɸ=hydrophobic)	Mg²⁺ coordination and catalysis
  C-motif (signature)	LSGGQ	Contacts ATP bound to opposite monomer
  D-loop	SALD	Connects ATP binding to conformational changes
  H-loop	H	Positions attacking water molecule
  Q-loop	Q	Coordinates Mg²⁺ and senses γ-phosphate

• Critical residues for catalysis:

  • Lysine in Walker A: coordinates β- and γ-phosphates

  • Aspartate in Walker B: coordinates Mg²⁺

  • Glutamate in Walker B: catalytic base for water activation

  • Histidine in H-loop: positions attacking water molecule

• Conformational changes during ATP binding and hydrolysis:

  • Closed dimer formation upon ATP binding

  • Rotation between domains during catalytic cycle

  • Signal transmission to transmembrane domains

How can researchers effectively study the dynamics between SsuB accessibility and protein production?

Understanding the relationship between mRNA accessibility at the translation initiation site and SsuB protein production requires integrating experimental and computational approaches:

• Experimental accessibility modulation:

  • Synonymous codon substitutions in the first 9 codons

  • Systematic alteration of predicted mRNA secondary structures

  • Measurement of expression levels from constructs with varying accessibility

• Computational modeling approaches:

  • Stochastic simulation modeling of translation initiation

  • Prediction of ribosome binding and translation rates

  • Analysis of mRNA folding dynamics using Boltzmann's ensemble calculations

  • Determination of opening energies (ΔGopen) for translation initiation sites

• Correlation analysis:

  • Relating computationally predicted accessibility to experimental expression levels

  • Identifying threshold values for successful expression

  • Determining the sensitivity of expression to incremental accessibility changes

• Integrated optimization strategy:

  • Design of sequences with tailored accessibility profiles

  • Predictive modeling of expression outcomes

  • Experimental validation and iterative refinement

  • Development of "expression tuning" rather than simple maximization

Stochastic simulation models have shown that higher accessibility leads to higher protein production but slower cell growth, which provides a framework for understanding how to balance expression efficiency with host cell physiology for optimal outcomes .

What emerging technologies might improve recombinant SsuB production and characterization in the near future?

Several cutting-edge technologies are poised to significantly advance recombinant SsuB research:

• Cell-free protein synthesis systems:

  • Elimination of cellular viability constraints

  • Direct access to reaction conditions for optimization

  • Rapid prototyping of expression constructs

  • Incorporation of non-canonical amino acids for functional studies

• CRISPR-Cas9 based host cell engineering:

  • Knockout of inhibitory host factors

  • Integration of supporting metabolic pathways

  • Creation of specialized chassis strains for ABC transporter expression

  • Genome-scale optimization of cellular resources

• Advanced structural biology techniques:

  • Cryo-EM for structure determination without crystallization

  • Single-molecule FRET for dynamic conformational studies

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

  • Integrative structural biology combining multiple data sources

• Artificial intelligence for expression optimization:

  • Machine learning algorithms predicting expression outcomes

  • Neural networks for sequence optimization

  • Automated experimental design and execution

  • Integration of multi-omics data for holistic understanding

These technologies promise to overcome current limitations in recombinant protein production and provide deeper insights into the structure-function relationships of SsuB and other ABC transporters .

How can researchers integrate SsuB functional studies with broader understanding of Acinetobacter species pathogenicity and resistance mechanisms?

Acinetobacter species, particularly A. baumannii, have emerged as significant nosocomial pathogens with alarming antibiotic resistance. Integrating SsuB studies within this broader context can yield valuable insights:

• Role in nutrient acquisition and survival:

  • SsuB involvement in alternative sulfur source acquisition

  • Contribution to survival in sulfate-limited environments

  • Potential role in host-pathogen interactions

  • Connection to metabolic adaptation during infection

• Relationships with virulence mechanisms:

  • Correlation with biofilm formation capabilities

  • Potential interplay with other virulence factors

  • Expression patterns during infection processes

  • Regulation in response to host environment stresses

• Connections to antimicrobial resistance:

  • Potential involvement in efflux of antimicrobial compounds

  • Co-regulation with other resistance mechanisms

  • Expression changes in response to antibiotic exposure

  • Contribution to persistence under antibiotic pressure

• Comparative analysis across clinical isolates:

  • Sequence and expression variation in diverse strains

  • Correlation of variants with clinical outcomes

  • Identification of strain-specific functional adaptations

  • Potential as a target for novel therapeutic approaches