Recombinant Archaeoglobus fulgidus Signal recognition particle 19 kDa protein (srp19)

What is the three-dimensional structure of Archaeoglobus fulgidus SRP19 and how does it compare to canonical RNA-binding motifs?

Archaeoglobus fulgidus SRP19 (Af-SRP19) is a 104-residue protein with a three-dimensional solution structure determined by NMR spectroscopy. The protein contains three beta-strands and two alpha-helical regions arranged in a betaalphabetabetaalpha topology, complemented by a 3(10) helix and a disordered C-terminal tail . This fold bears resemblance to the betaalphabetabetaalphabeta RNP motif common in RNA-binding proteins, which typically engage RNA through conserved sequence motifs within beta-strands 1 and 3.

What are the optimal protocols for recombinant expression and purification of Af-SRP19?

For efficient expression of recombinant Af-SRP19, the gene should be amplified from Archaeoglobus fulgidus genomic DNA using primers containing appropriate restriction sites (such as NdeI and HindIII) and inserted into a bacterial expression vector like pET23c . The construct should then be transformed into E. coli for protein expression.

The optimized purification protocol includes:

• Induction of protein expression with IPTG in transformed E. coli cultures

• Cell lysis and solubilization of Af-SRP19 in buffer containing 2 M urea

• Loading the solubilized protein onto a cation exchange column

• Gradual removal of urea through controlled buffer exchange

• Elution of purified protein at high salt concentration (approximately 1.9 M)

The purified protein typically migrates at ~15 kDa on SDS-PAGE despite its calculated molecular weight of 12,405 Da, likely due to its highly basic character (theoretical pKi = 11.05) . Successful purification yields active protein molecules as confirmed by their quantitative association with SRP RNA in binding assays .

How does Af-SRP19 contribute to archaeal SRP assembly and what is its relationship with SRP54?

Af-SRP19 plays a critical role in archaeal SRP assembly by promoting the association of SRP54 (the signal peptide-binding protein) with SRP RNA . Chemical modification studies using hydroxyl radicals and DEPC have revealed that Af-SRP19 and Af-SRP54 bind to non-overlapping primary sites on SRP RNA, with SRP19 binding to the tips of helix 6 and helix 8, while SRP54 associates with the distal loop and asymmetric bulge of helix 8 .

Importantly, Af-SRP19 binding induces significant conformational changes concentrated in the proximal asymmetric bulge of helix 8 . Selected nucleotides in this bulge become more susceptible to chemical modification following SRP19 binding but are subsequently protected when SRP54 joins the complex. This indicates that SRP19 binding reorganizes the RNA structure to create a high-affinity binding site for SRP54 .

Unlike eukaryotic SRP54, archaeal SRP54 demonstrates significant intrinsic affinity for SRP RNA even without SRP19 (KD approximately 15 nM) . This unique property has enabled researchers to directly compare particles formed with and without SRP19, providing precise insights into SRP19's role in the assembly process. The current model suggests that SRP19 bridges the ends of helix 6 and helix 8, inducing a long-range structural change that presents the proximal bulge in a conformation optimal for high-affinity SRP54 binding .

What are the molecular determinants of Af-SRP19 binding to SRP RNA?

The interaction between Af-SRP19 and SRP RNA involves specific structural elements and follows a distinct mechanism. Unlike typical RNA-binding proteins that recognize specific nucleotide sequences, Af-SRP19 primarily contacts the phosphate backbone of the RNA, particularly at the tetraloop regions of helices 6 and 8 .

Native gel mobility shift assays have shown that approximately 21 ng of protein is required to bind 200 ng of SRP RNA to achieve 50% binding, corresponding to an RNA/protein molar ratio of 0.86 . This suggests a nearly 1:1 stoichiometry in the complex formation.

The binding mechanism involves:

• Initial recognition of the tetraloop structures at the tips of helices 6 and 8

• Stabilization of the complex through interactions between residues in and around beta-strand 1 of Af-SRP19 and the phosphate backbone of the RNA

• Maintenance of an exposed orientation of the tetraloop bases, which differs from the base-specific recognition seen in canonical RNP motifs

• Inducing structural changes in the proximal asymmetric bulge of helix, creating a high-affinity binding site for SRP54

This binding mechanism appears relatively conserved across species, as Af-SRP19 can interact with SRP RNAs from different organisms, including Methanococcus jannaschii and human SRP RNAs, although with varying affinities .

What methodological approaches are most effective for analyzing Af-SRP19-RNA interactions?

Several complementary experimental approaches have proven effective for studying the interactions between recombinant Af-SRP19 and SRP RNA:

• DEAE Column Binding Assays: This method can verify complex formation by demonstrating that Af-SRP19 bound to SRP RNA is retained on a DEAE column in 300 mM KOAc buffer and elutes at 1 M KOAc, while free protein appears in the flowthrough .

• Native Polyacrylamide Gel Electrophoresis: This technique allows direct visualization of protein-RNA complexes through mobility shift analysis. By titrating increasing amounts of protein with a fixed amount of RNA, researchers can monitor complex formation and estimate binding parameters .

• Chemical Modification Studies: Methods employing hydroxyl radicals and DEPC have been instrumental in identifying precise binding sites and detecting conformational changes in the RNA upon protein binding . These approaches have revealed that SRP19 and SRP54 bind to non-overlapping sites and that SRP19 induces structural changes in regions that subsequently interact with SRP54 .

• Filter Binding Assays: Combined with gel mobility shift and Ni-NTA agarose bead binding assays, these methods allow determination of binding constants for binary and ternary complexes of SRP proteins and SRP RNA .

• Cross-species Binding Experiments: Comparing Af-SRP19 binding to SRP RNAs from different species provides insights into binding specificity and conservation of interaction mechanisms .

For comprehensive analysis, researchers should employ multiple techniques to overcome the limitations of individual methods and obtain robust, complementary data on binding affinities, stoichiometry, and structural changes.

How can researchers reconstitute functional archaeal SRP from recombinant components?

The in vitro reconstitution of archaeal SRP from recombinant components follows a systematic protocol:

• SRP RNA Preparation: Clone the SRP RNA gene under a T7 promoter using appropriate primers with restriction sites (e.g., EcoRI and BamHI). Generate the RNA through in vitro transcription with T7 RNA polymerase .

• RNA Renaturation: Properly fold the SRP RNA by heating in binding buffer to 65°C followed by slow cooling to room temperature .

• Component Assembly: Two approaches can be used:

  • Sequential addition: Add Af-SRP19 first to form a binary complex, followed by addition of Af-SRP54

  • Simultaneous addition: Mix both proteins with SRP RNA concurrently, which typically achieves complete binding of both proteins

• Verification of Assembly: Confirm successful reconstitution through:

  • DEAE column binding: Properly assembled SRP elutes at high salt concentrations

  • Native PAGE: Formation of shifted RNA-protein complexes

  • Functional assays: Test if the reconstituted SRP can associate with signal peptides, such as that of bovine pre-prolactin translated in vitro

A key finding from reconstitution studies is that archaeal SRP54, unlike its eukaryotic counterparts, exhibits significant intrinsic affinity for SRP RNA even without SRP19 . Nevertheless, SRP19 enhances SRP54 binding considerably, and complete binding of both proteins is most efficiently achieved when added simultaneously to the RNA .

The reconstituted archaeal SRP maintains its signal sequence recognition capability, indicating preservation of its core biological function in protein targeting .

What are the key similarities and differences between archaeal and eukaryotic SRP19 proteins?

Archaeal and eukaryotic SRP19 proteins share significant structural and functional features while exhibiting important differences:

Similarities:

• Both adopt similar folds resembling the RNP motif common in RNA-binding proteins

• Both bind to corresponding regions of their respective SRP RNAs (tips of helices 6 and 8)

• Both engage RNA primarily through contacts with the phosphate backbone rather than through base-specific interactions

• Both promote SRP54 association with SRP RNA by inducing conformational changes that facilitate binding

• Significant cross-reactivity exists, with archaeal SRP proteins capable of binding to eukaryotic SRP RNA and vice versa

Differences:

• SRP54 Dependency: Archaeal SRP54 has significant intrinsic affinity for SRP RNA even without SRP19 (KD ~15 nM) , whereas eukaryotic SRP54 binding is strictly dependent on SRP19

• Binding Specificity: The dependence on SRP19 for SRP54 binding is most pronounced with components from the same species

• Component Complexity: Archaeal SRP appears simpler, containing only homologs of SRP RNA, SRP19, and SRP54, while eukaryotic SRP includes additional components

• Structural Adaptations: Specific structural features have likely evolved to accommodate the different cellular environments and protein targeting requirements in archaea versus eukaryotes

These similarities and differences reflect both the evolutionary conservation of fundamental SRP assembly mechanisms and the specific adaptations that have occurred in different domains of life.

What is the binding specificity of Af-SRP19 for heterologous SRP RNAs and what does this reveal about SRP evolution?

Experiments examining the binding specificity of recombinant Af-SRP19 across different SRP RNAs provide valuable insights into SRP evolution:

When tested against SRP RNAs from Archaeoglobus fulgidus, Methanococcus jannaschii, and humans, Af-SRP19 demonstrated the ability to bind all three, indicating substantial cross-species compatibility . Native gel mobility shift assays revealed that when Af-SRP19 was added to a mixture containing these three different RNAs, the free RNA amounts decreased for all species, confirming interaction with each RNA .

Particularly noteworthy was the observation that M. jannaschii SRP RNA could bind to human SRP54M quantitatively even without SRP19 , which is unusual given that eukaryotic SRP54 typically requires SRP19 for RNA binding. This suggests that certain archaeal SRP RNAs may have intrinsic structural properties that allow direct interaction with SRP54.

These findings reveal that:

• The fundamental RNA recognition mechanisms are conserved across different domains of life

• Specific adaptations have occurred during SRP evolution in different lineages

• Archaeal SRP components may represent a more ancestral form with broader binding capabilities

• The co-evolution of SRP components has led to optimized interactions within species

How do SRP19-induced conformational changes in SRP RNA regulate SRP assembly?

The binding of Af-SRP19 to SRP RNA induces specific conformational changes that play a crucial regulatory role in SRP assembly. Chemical modification studies have provided detailed insights into these structural rearrangements:

When Af-SRP19 binds to the tips of helix 6 and helix 8 of SRP RNA, it induces conformational changes concentrated in the proximal asymmetric bulge of helix 8 . Specifically, certain nucleotides in this bulge become more susceptible to chemical modification (with DEPC and hydroxyl radicals) after SRP19 binding, indicating they undergo structural rearrangements or become more exposed .

This conformational change is functionally significant because these same nucleotides that become modified following SRP19 binding are subsequently protected from modification when SRP54 joins the complex . This sequential pattern of exposure followed by protection reveals a molecular mechanism for SRP assembly regulation:

• SRP19 binding acts as a molecular switch that alters RNA conformation

• This altered conformation creates a high-affinity binding site for SRP54

• SRP54 then directly interacts with the newly exposed or repositioned nucleotides

The current model suggests that SRP19 functions as a bridge between helices 6 and 8, inducing a long-range structural change that presents the proximal bulge of helix 8 in a conformation compatible with high-affinity SRP54 binding . This mechanism explains why SRP19 generally enhances SRP54 binding to SRP RNA, particularly in eukaryotic systems where the dependency is absolute.

This RNA conformational switching represents an elegant regulatory mechanism in which one protein component (SRP19) prepares the binding site for another protein component (SRP54) through allosteric effects on the RNA structure.

What are the key biochemical characteristics of recombinant Af-SRP19 and how do they impact experimental design?

Recombinant Af-SRP19 possesses several distinctive biochemical properties that researchers should consider when designing experiments:

• Molecular Weight and Electrophoretic Mobility: While the calculated molecular weight based on amino acid sequence is 12,405 Da, Af-SRP19 migrates at approximately 15 kDa on SDS-PAGE . This anomalous migration results from its highly basic character (theoretical pKi = 11.05) and should be accounted for when analyzing gel results .

• Solubility and Stability: During purification, Af-SRP19 initially requires solubilization in buffer containing 2 M urea, which must be gradually removed through controlled buffer exchange . This suggests potential solubility challenges that might necessitate careful buffer optimization in experimental setups.

• Thermostability: As a protein from a hyperthermophilic archaeon, Af-SRP19 likely possesses considerable thermal stability, potentially allowing experiments at elevated temperatures that might benefit studies of structure and binding kinetics.

• RNA Binding Properties: Af-SRP19 binds to SRP RNA with near 1:1 stoichiometry (RNA/protein molar ratio of 0.86 at 50% binding) . It forms stable complexes with SRP RNA detectable by methods such as DEAE column binding and native gel mobility shift assays .

• Structural Rigidity: The protein undergoes only minor structural changes upon RNA binding , suggesting pre-organization of its binding interface. This property may influence the thermodynamics and kinetics of complex formation.

• Cross-species Binding Capability: Af-SRP19 can bind to SRP RNAs from different species , offering opportunities for comparative studies but also necessitating careful controls when studying species-specific interactions.

These biochemical properties make Af-SRP19 a valuable model for studying RNA-protein interactions while requiring specific considerations for experimental design, including appropriate buffer conditions, temperature settings, and protein concentration ranges for binding studies.