Recombinant Bartonella quintana 30S ribosomal protein S16 (rpsP)

How does Bartonella quintana S16 compare structurally to other bacterial ribosomal S16 proteins?

Structural analysis of ribosomal proteins across species reveals important patterns:

• Secondary structure content: Human S16 contains approximately 21% α-helices and 24% β-strands . Bacterial S16 proteins, including B. quintana's, likely have similar secondary structure distributions.

• Stability characteristics: Like human S16, B. quintana S16 would be expected to show pH sensitivity, with denaturation occurring at pH values above 8.0. The protein would likely demonstrate gradual unfolding with increasing denaturant concentrations .

• Sequence conservation: Core functional domains that interact with 16S rRNA are typically highly conserved across bacterial species, while surface-exposed regions may show greater variability reflecting species-specific adaptations.

Comparison methods should include multiple sequence alignments, homology modeling based on existing crystal structures, and experimental verification through techniques like circular dichroism spectroscopy to assess secondary structure content.

What expression systems are most effective for producing recombinant B. quintana S16?

Based on experience with related proteins, several expression systems can be considered:

E. coli-based expression:

• pET vector systems, particularly pET-15b (used successfully for human S16), provide high-level expression with a histidine tag for purification

• BL21(DE3) or Rosetta strains help address potential codon usage bias

• Lower temperature expression (16-25°C) often improves solubility

• IPTG concentration should be optimized to prevent formation of inclusion bodies

Solubility enhancement strategies:

• Fusion partners such as SUMO, MBP, or GST may improve solubility

• Co-expression with molecular chaperones can assist proper folding

• Addition of solubility-enhancing agents like sorbitol or trehalose to growth media

Inclusion body recovery:
If the protein forms inclusion bodies (as observed with human S16), a structured refolding protocol is essential:

• Solubilize inclusion bodies using suitable denaturants (8M urea or 6M guanidine hydrochloride)

• Perform step-wise dialysis with gradually decreasing denaturant concentrations

• Include redox couples (reduced/oxidized glutathione) to assist disulfide bond formation if applicable

• Monitor refolding by CD spectroscopy to verify secondary structure formation

What are the optimal purification strategies for recombinant B. quintana S16 protein?

A multi-step purification strategy is recommended:

Initial capture:

• Immobilized metal affinity chromatography (IMAC) using His-tag fusion is the preferred first step

• Ni-NTA or Co-NTA resins provide high affinity and capacity

• Include low concentrations of imidazole (10-20 mM) in binding buffers to reduce non-specific binding

Intermediate purification:

• Ion exchange chromatography (typically cation exchange as ribosomal proteins are generally basic)

• Heparin affinity chromatography can be particularly useful for RNA-binding proteins

Polishing steps:

• Size exclusion chromatography to remove aggregates and ensure homogeneity

• Reverse-phase HPLC for highest purity requirements

Buffer optimization:

• Maintain pH between 6.0-7.5 based on stability studies of related proteins

• Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

• Consider stabilizing additives like glycerol or arginine to prevent aggregation

Quality control should include SDS-PAGE, Western blotting, mass spectrometry for identity confirmation, and functional assays such as RNA binding tests.

How can researchers assess the functional activity of purified recombinant B. quintana S16?

Functional assessment requires multiple complementary approaches:

Structural integrity:

• Circular dichroism (CD) spectroscopy to verify secondary structure content

• Thermal shift assays to assess protein stability

• Size exclusion chromatography to confirm monomeric state

RNA binding capability:

• Electrophoretic mobility shift assays (EMSA) with 16S rRNA fragments

• Filter binding assays for quantitative binding affinity measurements

• Surface plasmon resonance (SPR) for detailed binding kinetics

Functional reconstitution:

• In vitro reconstitution of partial 30S subunits with defined components

• Hydroxyl radical footprinting to assess protection patterns similar to those observed in E. coli

• Translation assays using reconstituted ribosomes containing the recombinant S16

A comprehensive functional assessment would combine these approaches to verify that the recombinant protein displays native-like structural and binding properties.

How can researchers monitor S16-mediated conformational changes during ribosome assembly?

Monitoring conformational changes requires sophisticated biophysical techniques:

Hydroxyl radical footprinting:
This technique has been successfully employed with E. coli S16 to reveal how it influences rRNA structure during assembly . The approach can:

• Identify which rRNA regions become protected upon S16 binding

• Detect conformational changes in distant regions (like helix 3) affected by S16 binding

• Monitor assembly intermediates by time-resolved experiments

FRET-based approaches:

• Site-specific labeling of S16 and its interaction partners with fluorophores

• Real-time monitoring of distance changes during assembly

• Single-molecule FRET to capture assembly heterogeneity

Structural methods:

• Cryo-electron microscopy of assembly intermediates

• Time-resolved small-angle X-ray scattering (TR-SAXS)

• NMR spectroscopy with isotopically labeled proteins

Experimental strategy should include:

• In vitro reconstitution of defined components

• Time-resolved measurements using complementary techniques

• Correlation of structural changes with functional outcomes

• Comparison with assembly patterns observed in E. coli

What strategies can overcome challenges in producing functional B. quintana S16?

Production of functional recombinant S16 presents several challenges:

Expression challenges:

• Potential toxicity to host cells due to interference with host ribosome assembly

• Inclusion body formation requiring complex refolding procedures

• Codon bias between B. quintana and expression hosts

Stability issues:

• Rapid denaturation at pH above 8.0, similar to human S16

• Susceptibility to proteolytic degradation

• Aggregation propensity in the absence of binding partners

Solutions table:

Systematic optimization of these parameters is essential for successful production of functional protein.

How does B. quintana S16 interact with other components of the 30S ribosomal subunit?

The interactions of S16 within the ribosome can be analyzed at multiple levels:

rRNA interactions:
Based on E. coli studies, S16 likely binds primarily to helices 15 and 17 in 16S rRNA . Key features include:

• The protein likely straddles a C-loop motif in helix 15 that stacks against bases in helix 17

• Binding of S16 stabilizes tertiary interactions between these helices

• S16 binding reduces the magnesium requirement for these interactions from 4.9 to 2.4 mM MgCl₂

Protein-protein interactions:

• S16 binding depends on primary assembly proteins S4 and S20 in E. coli

• These proteins pre-organize the S16 binding site on the rRNA

• S16 likely participates in cooperative binding networks with other ribosomal proteins

Long-range effects:
One of the most significant findings about S16 is its ability to influence distant regions of the ribosome:

• In E. coli, S16 binding to helices 15/17 triggers a conformational switch at helix 3 approximately 30Å away

• This conformational change stabilizes pseudoknots in the decoding center

• The protein also stabilizes tertiary contacts with helix 6/6a in the core of the 5′ domain

Methods to characterize these interactions include chemical crosslinking, hydrogen-deuterium exchange mass spectrometry, and in vitro reconstitution with defined components.

What mutagenesis approaches can be used to study structure-function relationships in B. quintana S16?

Multiple mutagenesis strategies can provide insights into S16 function:

Site-directed mutagenesis:

• Target conserved residues identified through sequence alignments

• Focus on potential RNA-binding residues (basic and aromatic amino acids)

• Create alanine substitutions to identify essential residues

• Studies in E. coli have highlighted the importance of Tyr17, which donates a hydrogen bond to the 2′ OH of A374 in the C-loop of helix 15

Domain-swapping:

• Replace domains of B. quintana S16 with corresponding regions from other species

• Create chimeric proteins to identify species-specific functional elements

In vivo validation:
B. quintana mutagenesis systems have been developed, as demonstrated with the hbpA gene:

• Transformation-competent strains can be prepared by electroporation

• Suicide vectors containing internal fragments of the target gene can be used

• Selection on appropriate antibiotics identifies successful mutants

A systematic approach would:

• Generate mutations based on structural predictions

• Express and characterize mutant proteins in vitro

• Test effects on RNA binding and ribosome assembly

• Validate findings using in vivo systems where possible

How can recombinant B. quintana S16 be used to investigate potential antimicrobial targets?

Ribosomal protein S16 offers several avenues for antimicrobial development:

Ribosome assembly inhibition:

• S16 plays a critical role in suppressing non-native assembly intermediates

• Compounds that interfere with this function could prevent proper ribosome formation

• High-throughput screening of compound libraries against recombinant S16 could identify potential inhibitors

Decoding center targeting:

• S16 stabilizes pseudoknots in the 30S decoding center that are essential for protein synthesis

• This connection to the decoding center suggests that S16 function may indirectly affect aminoglycoside binding

• Structure-based design could target the interface between S16 and critical rRNA elements

Experimental approaches:

• Develop biochemical assays for S16 function (RNA binding, conformational changes)

• Screen compound libraries for inhibitors of these functions

• Characterize hits using structural and functional studies

• Assess specificity by comparing effects on human versus bacterial S16 proteins

• Validate promising compounds in cellular infection models

Targeting assembly factors like S16 represents a novel approach that could circumvent existing resistance mechanisms, as these proteins are not directly targeted by current antibiotics.