Recombinant Enterobacteria phage RB3 Single-stranded DNA-binding protein (32)

What are the structural features of RB3 SSB and how do they compare to other phage SSBs?

RB3 SSB contains a core single-stranded DNA binding domain and a C-terminal regulatory region similar to other enterobacteria phage SSBs. The C-terminal region appears to play a crucial regulatory role in DNA binding. Analysis of E. coli SSB shows that the last 8 highly acidic amino acids in the C-terminal tails can inhibit ssDNA binding, particularly at moderate salt concentrations . This inhibitory effect decreases as salt concentration increases, which likely applies to RB3 SSB given structural similarities observed across phage SSBs. For structural studies, techniques like SEC-MALLS (Size Exclusion Chromatography coupled to Multiangle Laser Light Scattering) can be employed as demonstrated with other recombinant proteins .

How does RB3 SSB function in DNA replication and repair processes?

RB3 SSB binds to single-stranded DNA regions created during DNA replication, recombination, and repair processes, protecting these vulnerable regions from nuclease degradation and preventing secondary structure formation. Similar to other SSBs, RB3 likely exhibits a binding mechanism where initial binding to approximately half of the protein subunits relieves the inhibitory effect for all subunits . This cooperative binding behavior is critical for efficient coating of ssDNA during these processes. For functional studies, researchers should consider varying salt concentrations in their experimental design, as binding properties of SSBs can be significantly salt-dependent .

What methods are recommended for initial functional testing of purified RB3 SSB?

For initial functional testing, electrophoretic mobility shift assays (EMSAs) using short oligodeoxynucleotides can determine basic binding properties. When designing these experiments, researchers should test binding at various salt concentrations (50-300 mM NaCl) to characterize salt-dependent binding behavior . Fluorescence anisotropy with labeled ssDNA is another valuable approach to quantify binding affinity and kinetics. To detect protein-DNA interactions more precisely, researchers can employ ultracentrifugation or size exclusion chromatography techniques similar to those used in analyzing tubulin-protein complexes .

What expression systems are most effective for producing recombinant RB3 SSB?

E. coli expression systems (particularly BL21 DE3 Star strains) are recommended for RB3 SSB production. Using a pET28 plasmid system with kanamycin resistance and IPTG-inducible promoter typically yields good results. Consider these protocol details:

• Express in LB medium supplemented with appropriate antibiotics

• Induce with 0.5 mM IPTG

• Maintain expression for 3 hours at 37°C

For initial protein extraction, nucleic acid precipitation using spermine followed by standard chromatography techniques is an effective approach . Expression levels can be monitored by SDS-PAGE, with expected bands corresponding to the predicted molecular weight of RB3 SSB.

What purification strategy yields the highest purity and activity for RB3 SSB?

A multi-step purification strategy is recommended:

• Initial clarification: Precipitate nucleic acids with spermine as demonstrated with other recombinant proteins

• Affinity chromatography: If using His-tagged constructs, employ Ni-NTA chromatography

• Ion exchange chromatography: Given the acidic C-terminal region typical of SSBs, anion exchange chromatography is effective for separating full-length protein from truncated variants

• Size exclusion chromatography: Final polishing step to ensure oligomeric homogeneity

Protein purity should be assessed by SDS-PAGE and activity through DNA binding assays. The presence of acidic C-terminal domains may influence chromatographic behavior, so buffer optimization is essential for preserving both structure and function.

How can researchers verify the oligomeric state of purified RB3 SSB?

To determine the oligomeric state of purified RB3 SSB, size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) provides accurate molecular weight determination in solution . This technique is particularly valuable for distinguishing between different oligomeric states. Analytical ultracentrifugation can provide complementary data on both size and shape. Native gel electrophoresis offers a simpler alternative but with lower resolution. When analyzing oligomeric state, researchers should consider buffer conditions carefully, as salt concentration may influence oligomerization.

How does the C-terminal domain of RB3 SSB influence its DNA binding properties?

The C-terminal domain, particularly its acidic residues, plays a critical regulatory role in ssDNA binding. Similar to E. coli SSB, the C-terminal region of RB3 SSB likely exerts an inhibitory effect on ssDNA binding, especially at moderate salt concentrations . To investigate this experimentally:

• Compare binding affinities of full-length RB3 SSB versus C-terminally truncated variants

• Assess binding at different salt concentrations (50-300 mM NaCl)

• Examine changes in cooperative binding behavior after C-terminal modification

Research on E. coli SSB shows that removal of the acidic C-terminal ends increases intrinsic affinity for ssDNA and enhances negative cooperativity between binding sites . This inhibitory effect diminishes at higher salt concentrations, and binding of ssDNA to approximately half of the SSB subunits relieves this inhibition for all subunits. These principles likely apply to RB3 SSB and should inform experimental design.

What biophysical techniques can elucidate binding mechanisms between RB3 SSB and ssDNA?

Multiple complementary biophysical techniques can provide insights into RB3 SSB-ssDNA interactions:

• Isothermal titration calorimetry (ITC): Determines thermodynamic parameters (ΔH, ΔS, ΔG) and binding stoichiometry

• Surface plasmon resonance (SPR): Measures association/dissociation kinetics in real-time

• Fluorescence spectroscopy: Using intrinsic tryptophan fluorescence or labeled DNA to monitor binding

• Circular dichroism (CD): Detects conformational changes upon binding

For rigorous mechanistic studies, combining these approaches with varying DNA lengths, sequences, and solution conditions provides the most comprehensive understanding. Structural determination methods like X-ray crystallography can be pursued using approaches similar to those employed for other DNA-binding proteins, where rigid body refinement treating protein domains separately has proven effective .

How can researchers quantify the cooperativity of RB3 SSB binding to ssDNA?

Quantifying cooperativity in RB3 SSB binding requires specialized experimental approaches:

• Multiple titration experiments: Conduct DNA binding titrations at varying protein concentrations

• Hill plot analysis: Calculate Hill coefficients to determine positive or negative cooperativity

• McGhee-von Hippel analysis: Apply this model to determine binding site size and cooperativity parameters

• Single-molecule techniques: FRET or optical tweezers can directly visualize cooperative binding events

The expected pattern based on E. coli SSB studies suggests that removal of the C-terminal domain enhances negative cooperativity between ssDNA binding sites . Researchers should design experiments that can distinguish between different binding modes (e.g., 35-nucleotide binding mode vs. 65-nucleotide binding mode) that may exist under different solution conditions.

How can RB3 SSB be utilized in DNA amplification and sequencing applications?

RB3 SSB can enhance DNA amplification and sequencing through several mechanisms:

• PCR enhancement: Addition of RB3 SSB can improve amplification of GC-rich or secondary structure-prone templates by preventing hairpin formation

• Isothermal amplification: In methods like RPA or LAMP, RB3 SSB stabilizes primer-template junctions and facilitates strand displacement

• Sequencing applications: Can improve read length and accuracy by minimizing secondary structure formation

When optimizing these applications, researchers should determine the optimal protein-to-DNA ratio and consider how the salt concentration affects binding properties. Testing truncated variants lacking the C-terminal domain may prove beneficial in applications requiring stronger DNA binding under specific conditions, as suggested by studies of E. coli SSB C-terminal modifications .

What role can RB3 SSB play in studying DNA repair mechanisms?

RB3 SSB can serve as a valuable tool for studying DNA repair mechanisms:

• In vitro reconstitution: Use purified RB3 SSB to reconstitute repair pathways like homologous recombination

• SSB-interactome studies: Identify and characterize proteins that interact with RB3 SSB using pull-down assays

• Single-molecule approaches: Visualize repair processes in real-time using fluorescently labeled RB3 SSB

The protein purification approaches used for stathmin-like domain proteins, including nucleic acid precipitation with spermine followed by chromatography, can be adapted for producing RB3 SSB for these studies . When designing experiments, researchers should account for the regulatory effects of the C-terminal domain on DNA binding, as this domain typically mediates protein-protein interactions in SSB proteins .

How can researchers use RB3 SSB to study phage-host interactions?

RB3 SSB provides insights into phage-host interactions through several research approaches:

• Host range studies: Compare RB3 SSB activity with host SSBs to identify functional similarities and differences

• Competition assays: Assess whether RB3 SSB can compete with host SSB for binding to ssDNA or host proteins

• Co-immunoprecipitation: Identify host proteins that interact with RB3 SSB during infection

Such studies could reveal mechanisms similar to those observed with phage CBB, which demonstrates broad host range characteristics across multiple bacterial genera like Pectobacterium, Erwinia, and Cronobacter . For host range studies, researchers should establish standardized plaque assays using 0.2% agarose overlays rather than standard agar overlays for more consistent plaque formation, as demonstrated effective with other enterobacteria phages .

How can researchers address solubility issues when expressing recombinant RB3 SSB?

Solubility challenges with RB3 SSB can be addressed through several strategies:

• Expression temperature optimization: Lower induction temperature to 18-25°C

• Co-expression with chaperones: Consider co-expressing with GroEL/GroES system

• Fusion tags: Test solubility enhancement tags like MBP, SUMO, or Thioredoxin

• Buffer optimization: Increase salt concentration (250-500 mM NaCl) and add stabilizers like glycerol (10-15%)

When purifying the protein, omitting the heating step used in some protocols may be necessary to prevent aggregation, as has been observed with other recombinant proteins like R4 and R4a . For challenging constructs, analyzing the predicted structure to identify hydrophobic patches that might contribute to aggregation can guide rational design of more soluble variants.

What are common pitfalls in interpreting RB3 SSB binding data and how can they be avoided?

Several factors can complicate interpretation of RB3 SSB binding data:

• Protein aggregation: Verify protein monodispersity through DLS or SEC before binding studies

• DNA secondary structures: Use well-characterized DNA oligonucleotides with minimal secondary structure

• Buffer effects: Salt concentration significantly affects SSB binding properties; standardize and report all buffer components

• Cooperative binding effects: Binding may not follow simple 1:1 models; apply appropriate cooperative binding models

Researchers should include proper controls, such as heat-denatured protein and non-specific DNA sequences. When comparing binding properties of different constructs (e.g., full-length versus truncated proteins), ensure equimolar active protein concentrations by first determining the fraction of active protein through stoichiometric binding assays with excess DNA.

How can researchers distinguish between specific and non-specific DNA binding by RB3 SSB?

Distinguishing specific from non-specific binding requires carefully designed experiments:

• Competition assays: Compare displacement by specific versus non-specific competitors

• Salt titration experiments: Specific interactions typically display greater salt resistance

• DNA length dependence: Characterize binding to oligonucleotides of different lengths

• Binding kinetics: Specific interactions often show different association/dissociation kinetics

Based on studies of E. coli SSB, researchers should expect salt-dependent binding behavior with inhibitory effects from the C-terminal domain being most pronounced at moderate salt concentrations . Experimental designs should account for these effects when distinguishing binding modes.

What crystallization strategies are most effective for RB3 SSB-DNA complexes?

Crystallization of RB3 SSB-DNA complexes presents several challenges that can be addressed through strategic approaches:

• Construct optimization: Test C-terminally truncated variants that remove flexible regions

• DNA design: Optimize oligonucleotide length and sequence to promote crystal packing

• Complex formation: Pre-form and purify the protein-DNA complex before crystallization

• Crystallization conditions: Screen various precipitants, focusing on conditions with moderate to high salt

For structure determination, molecular replacement using related SSB structures as search models is likely to be effective. Rigid body refinement, in which protein domains are refined separately, has proven successful with other DNA-binding proteins . When analyzing diffraction data, watch for increased correlation coefficients as more protein subunits are found during molecular replacement, similar to patterns observed with other oligomeric protein-DNA complexes .

How can cryo-EM be applied to study the structural dynamics of RB3 SSB-DNA complexes?

Cryo-EM offers advantages for studying dynamic RB3 SSB-DNA complexes:

• Sample preparation: Optimize grid type, protein concentration, and freezing conditions

• Data collection strategy: Collect tilt series for tomography to visualize binding along DNA strands

• Image processing: Apply 3D classification to identify different binding modes

• Structural analysis: Compare with crystal structures to identify conformational changes

This approach is particularly valuable for visualizing how RB3 SSB coats longer ssDNA substrates and undergoes conformational changes during binding. The methodology used to visualize phage CBB's unique structural features through electron microscopy can inform approaches to studying RB3 SSB-DNA complexes .

How can researchers design experiments to investigate the effect of RB3 SSB phosphorylation on its activity?

To study potential phosphorylation effects on RB3 SSB:

• Site identification: Use bioinformatics to predict potential phosphorylation sites

• Site-directed mutagenesis: Create phosphomimetic (S/T→D/E) and phospho-null (S/T→A) mutants

• In vitro phosphorylation: Use purified kinases to generate phosphorylated protein

• Functional comparison: Compare DNA binding properties of phosphorylated versus non-phosphorylated protein

Drawing parallels from stathmin proteins like RB3, which are regulated by phosphorylation and affect microtubule dynamics , researchers should consider that phosphorylation might similarly regulate SSB activity through conformational changes. Analytical techniques similar to those used to characterize RB3 stathmin variants can be applied to study phosphorylated SSB variants .

What approaches can detect and characterize protein-protein interactions involving RB3 SSB?

Multiple complementary approaches can identify RB3 SSB interaction partners:

• Pull-down assays: Use tagged RB3 SSB to capture binding partners from cell lysates

• Yeast two-hybrid screening: Identify direct protein interactions

• Cross-linking mass spectrometry: Map interaction interfaces at amino acid resolution

• Fluorescence-based interaction assays: FRET or fluorescence complementation to verify interactions in vitro or in vivo

When designing these experiments, consider that SSB interactions are often mediated by the C-terminal acidic region, as is the case with E. coli SSB . Controls should include constructs lacking this domain to confirm its role in mediating specific protein-protein interactions.

How can single-molecule techniques advance our understanding of RB3 SSB function?

Single-molecule approaches provide unique insights into RB3 SSB activity:

• Single-molecule FRET: Directly observe conformational changes during DNA binding

• DNA curtains: Visualize movement and binding of fluorescently labeled RB3 SSB along DNA

• Optical tweezers: Measure forces involved in SSB-mediated DNA unwinding

• Super-resolution microscopy: Track RB3 SSB dynamics in reconstituted systems

These techniques can reveal transient intermediate states and heterogeneous behaviors masked in bulk experiments. Methodological approaches similar to those used to observe microtubule dynamics with dark field microscopy can be adapted to study RB3 SSB-DNA interactions at the single-molecule level.