Recombinant Synechocystis sp. Cysteine--tRNA ligase (cysS)

What is the function of Cysteine-tRNA ligase (cysS) in Synechocystis sp.?

Cysteine-tRNA ligase (cysS) belongs to the aminoacyl-tRNA synthetase family of enzymes that catalyze the attachment of cysteine to its cognate tRNA^Cys. In Synechocystis sp., as in other organisms, cysS plays a critical role in protein biosynthesis by ensuring the correct incorporation of cysteine into growing polypeptide chains. The enzyme typically performs a two-step reaction: first activating cysteine with ATP to form cysteinyl-AMP, then transferring the activated cysteine to the 3' end of tRNA^Cys, forming cysteinyl-tRNA^Cys.

While the direct aminoacylation pathway is common in most bacteria, including cyanobacteria, some organisms (particularly certain archaea) utilize an indirect pathway for cysteine incorporation. In this indirect pathway, O-phosphoserine is first attached to tRNA^Cys by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to cysteine by Sep-tRNA:Cys-tRNA synthase (SepCysS) while still bound to tRNA . This alternative mechanism represents an evolutionary adaptation that may reflect how cysteine was originally incorporated into the genetic code.

How does the cysteine biosynthesis pathway in Synechocystis sp. compare to other organisms?

Cysteine biosynthesis pathways vary significantly across different domains of life. Based on available research, there are two major pathways for cysteine-tRNA synthesis:

• Direct pathway: This is the conventional pathway found in most bacteria and eukaryotes, where cysteine is directly attached to tRNA^Cys by cysS.

• Indirect pathway: Found primarily in methanogens and some other archaea, this pathway involves the initial attachment of O-phosphoserine to tRNA^Cys by SepRS, followed by conversion to cysteine by SepCysS while still bound to tRNA.

In ancestral methanogens, a third protein, SepCysE, forms a bridge between SepRS and SepCysS to create a ternary complex called the transsulfursome. This complex enables "a global long-range channeling of tRNA^Cys between SepRS and SepCysS distant active sites" . This channeling mechanism facilitates the consecutive reactions of the two-step indirect pathway and may reflect the evolutionary mechanism by which cysteine was originally added to the genetic code .

While the search results don't specifically confirm which pathway Synechocystis sp. uses, cyanobacteria typically employ the direct pathway using cysS, consistent with their bacterial lineage.

What structural features characterize Synechocystis sp. cysS?

Although the search results don't provide specific structural information about Synechocystis sp. cysS, aminoacyl-tRNA synthetases typically share several conserved structural features that would likely be present:

• An aminoacylation domain containing the HIGH and KMSKS signature motifs responsible for ATP binding and amino acid activation

• An anticodon-binding domain that ensures specificity for tRNA^Cys

• Potentially an editing domain to prevent misacylation of tRNA^Cys with similar amino acids

The active site of cysS likely contains conserved cysteine residues that play crucial roles in enzyme function, similar to how the cysteine residues in the UirS protein of Synechocystis are essential for its photochemical properties . Mutagenesis studies of UirS demonstrated that "substitution of the blue/green CBCR subfamily-specific Cys533 ablated normal photochemistry and reduced but did not abolish chromophore binding" , illustrating how specific residues can have distinct functional roles.

Similar structural analysis and mutagenesis approaches could reveal the critical residues in cysS that determine its substrate specificity and catalytic activity.

How is cysS gene expression regulated in Synechocystis sp.?

The regulation of cysS gene expression in Synechocystis sp. likely integrates multiple environmental and metabolic signals to coordinate protein synthesis with cellular needs. While the search results don't provide direct information about cysS regulation, insights can be drawn from known regulatory mechanisms in Synechocystis.

In cyanobacteria, gene expression is often regulated in response to environmental conditions such as light intensity, nutrient availability, and various stresses. For example, Synechocystis sp. PCC6803 possesses a complex UV-A activated signaling system involving the cyanobacteriochrome UirS and response regulators UirR and LsiR that regulates negative phototaxis . This system demonstrates how Synechocystis can respond to environmental cues through sophisticated signal transduction pathways.

What are the optimal conditions for expressing recombinant Synechocystis sp. cysS in E. coli?

Optimizing the expression of recombinant Synechocystis sp. cysS in E. coli requires careful consideration of multiple parameters. Based on successful expression of other Synechocystis genes, the following conditions might be applicable:

The pTYB21 expression vector, which was successfully used for expressing the Synechocystis PCC6803 sll1621 gene , contains an IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system. This system allows for the purification of the target protein without remaining affinity tags, which can be advantageous for enzymatic studies where tags might interfere with activity.

Additional considerations for cysS expression include codon optimization for E. coli, as cyanobacterial codon usage differs from E. coli, and potential co-expression with molecular chaperones to improve protein folding. The presence of rare codons in the Synechocystis cysS sequence might necessitate the use of specialized E. coli strains like Rosetta that supply additional tRNAs for rare codons.

How can site-directed mutagenesis be used to study the active site of Synechocystis sp. cysS?

Site-directed mutagenesis represents a powerful approach to investigate the structure-function relationships in cysS. Taking inspiration from mutagenesis studies of other Synechocystis proteins, such as UirS where "Mutagenesis studies have established two conserved Cys residues to be required for the blue/green photocycles" , similar approaches can be applied to cysS.

For cysS, site-directed mutagenesis could target:

• Conserved residues in the HIGH and KMSKS motifs that are involved in ATP binding and amino acid activation

• Residues predicted to interact with the cysteine substrate

• Residues in the anticodon-binding domain that recognize tRNA^Cys

• Cysteine residues that might be involved in enzyme function or stability

The impact of these mutations can be assessed through multiple assays:

• Aminoacylation assays measuring the rate of cysteine attachment to tRNA^Cys

• ATP-PPi exchange assays to monitor the first step of the reaction (amino acid activation)

• Binding assays to determine changes in affinity for tRNA or amino acid substrates

• Thermal stability assays to assess effects on protein folding and stability

The results would provide insights into the catalytic mechanism and substrate specificity determinants of Synechocystis sp. cysS. For example, mutations that affect ATP binding might impact the first step of the reaction while preserving tRNA binding, whereas mutations in the anticodon-binding domain might specifically disrupt tRNA recognition.

What are the challenges in purifying recombinant Synechocystis sp. cysS with preserved enzymatic activity?

Purifying recombinant aminoacyl-tRNA synthetases with preserved activity presents several challenges that require careful optimization:

• Maintaining protein solubility: Aminoacyl-tRNA synthetases can aggregate when overexpressed, particularly in heterologous systems. Optimization of expression conditions (temperature, induction parameters) and buffer components (salt concentration, pH, additives like glycerol) is crucial to maintain the protein in a soluble, active form.

• Preserving native conformation: The enzymatic activity of cysS depends on its proper folding. Harsh purification conditions might disrupt the tertiary structure and lead to activity loss. Gentle purification methods and the inclusion of stabilizing agents like glycerol or reducing agents might help preserve activity.

• Removing affinity tags without affecting function: If using tagged recombinant cysS for purification, tag removal might be necessary to ensure native activity. The IMPACT system used with the pTYB21 vector mentioned in search result offers a potential solution, as it allows for tag-free protein purification through intein-mediated cleavage.

• Preventing proteolytic degradation: Addition of protease inhibitors during purification and working at cold temperatures can help maintain protein integrity by reducing protease activity.

• Ensuring cofactor retention: If cysS activity depends on metal ions or other cofactors, these should be included in purification buffers. Many aminoacyl-tRNA synthetases require divalent metal ions (typically Mg²⁺ or Zn²⁺) for catalytic activity.

Each of these challenges requires systematic optimization, often necessitating multiple rounds of expression and purification trials with varying conditions to achieve the highest yield of active enzyme.

How can the specificity of Synechocystis sp. cysS for tRNA^Cys be experimentally determined?

The specificity of cysS for its cognate tRNA^Cys is crucial for translational fidelity. Several experimental approaches can be used to investigate this specificity:

• In vitro aminoacylation assays:

  • Purified recombinant cysS can be incubated with various tRNA isoacceptors and radiolabeled cysteine

  • The amount of cysteine attached to each tRNA can be measured to determine specificity

  • Kinetic parameters (Km, kcat) for different tRNAs can be compared to quantify specificity differences

• Structural biology approaches:
  Similar to the approaches used to study the transsulfursome , multiple complementary techniques can provide insights into cysS-tRNA^Cys interactions:

  • X-ray crystallography of cysS-tRNA^Cys complex to reveal atomic details of recognition

  • Small-angle X-ray scattering (SAXS) to study complex formation in solution

  • Electron microscopy to visualize larger complexes

• Binding assays:

  • Electrophoretic mobility shift assays (EMSA) to detect cysS-tRNA binding

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Fluorescence anisotropy using labeled tRNA to monitor binding

• Competition experiments:

  • Using various tRNAs to compete with tRNA^Cys for binding to cysS

  • Determining which tRNA features are recognized by cysS

These approaches would provide complementary information about the structural basis of tRNA recognition by cysS and the kinetic consequences of this recognition for aminoacylation activity.

What experimental designs are most appropriate for studying the kinetics of Synechocystis sp. cysS?

Studying the kinetics of cysS requires rigorous experimental designs that allow for reliable determination of reaction rates and mechanisms. While single-case experimental designs (SCEDs) mentioned in search result are primarily discussed in the context of behavioral research, the principles of rigorous experimental design apply equally to biochemical studies.

For studying cysS kinetics, appropriate experimental designs include:

As noted in search result , experimental control and replication are essential: "Experimental control in SCEDs includes replication of the effect either within or between participants" . Similarly, biochemical studies should include appropriate controls and multiple replicates to ensure reliability and reproducibility of the results.

How can cysS activity be reliably measured in vitro?

Measuring cysS activity reliably requires selecting appropriate assay methods based on the specific research questions and available resources. Several established methods can be used:

• Radioactive assays:

  • Using ¹⁴C or ³H-labeled cysteine to track aminoacylation

  • Acid precipitation of aminoacylated tRNA followed by scintillation counting

  • TCA precipitation on filter papers followed by washing and counting

  • These methods offer high sensitivity but require radioactive materials handling

• Non-radioactive methods:

  • HPLC-based detection of aminoacylated vs. non-aminoacylated tRNA

  • Colorimetric assays measuring pyrophosphate release during amino acid activation

  • Mass spectrometry to detect aminoacylated tRNA products

  • These methods avoid radioactivity but may have lower sensitivity

• Real-time assays:

  • Coupled enzyme assays that link pyrophosphate release to NADH oxidation

  • Fluorescence-based assays using labeled tRNA or amino acid analogs

  • These allow continuous monitoring but may be affected by interfering reactions

For all methods, proper controls are essential, including:

• No-enzyme controls to account for spontaneous reactions

• Heat-inactivated enzyme controls

• Controls with inhibitors or incorrect substrates to verify specificity

• Internal standards for quantification

The choice of method should consider factors such as sensitivity requirements, available equipment, safety considerations, and the specific aspects of cysS activity being investigated.

What approaches can be used to investigate the relationship between cysS structure and function?

Understanding the structure-function relationship of cysS requires integrating multiple experimental approaches:

• X-ray crystallography:

  • Determining the atomic structure of cysS alone and in complex with substrates

  • Analyzing how mutations affect protein structure

  • Identifying conformational changes during catalysis

  • This provides the highest resolution structural information but requires protein crystallization

• Molecular dynamics simulations:

  • Modeling the dynamic behavior of cysS during substrate binding and catalysis

  • Predicting the effects of mutations on protein stability and function

  • Identifying potential allosteric regulation sites

  • These computational approaches complement experimental data by providing dynamic information

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Mapping regions of the protein that undergo conformational changes upon substrate binding

  • Identifying dynamic regions that might be important for catalysis

  • This technique provides information about protein dynamics in solution

• Structure-guided mutagenesis:
  Similar to the approach described in search result : "Mutagenesis of Cys561... resulted in the loss of PCB binding... By comparison, substitution of the blue/green CBCR subfamily-specific Cys533 ablated normal photochemistry and reduced but did not abolish chromophore binding"

  For cysS, structure-guided mutagenesis could:

  • Target residues predicted to be involved in substrate binding or catalysis

  • Create chimeric proteins to identify domains responsible for specific functions

  • Introduce disulfide bonds to restrict conformational changes

Combining these approaches provides a comprehensive understanding of how cysS structure determines its function, enabling rational design of variants with altered properties for research or biotechnological applications.

How can the in vivo role of cysS in Synechocystis sp. be investigated?

Investigating the in vivo role of cysS requires approaches that connect molecular function to cellular physiology:

• Gene knockout or knockdown strategies:

  • CRISPR-Cas9 mediated gene editing to create conditional cysS mutants

  • Inducible antisense RNA to temporarily reduce cysS expression

  • Analysis of resulting phenotypes (growth rate, protein synthesis patterns, stress responses)

  • As cysS is likely essential, conditional approaches would be necessary

• Complementation studies:

  • Expressing cysS from other organisms in Synechocystis sp. cysS mutants

  • Determining which features are conserved and which are species-specific

  • Testing mutant versions of cysS for their ability to complement function

• Protein-protein interaction studies:

  • Co-immunoprecipitation to identify cysS-interacting proteins

  • Yeast two-hybrid or bacterial two-hybrid screens

  • Proximity labeling methods (BioID, APEX) to identify proteins in close proximity to cysS in vivo

  • These approaches could reveal connections to other cellular processes

• Localization studies:

  • Fluorescent protein fusions to determine subcellular localization of cysS

  • Immunogold electron microscopy for high-resolution localization

  • Co-localization with other components of the translation machinery

• Systems biology approaches:

  • Transcriptomics to identify genes co-regulated with cysS

  • Proteomics to detect changes in protein expression when cysS function is altered

  • Metabolomics to identify changes in cysteine and related metabolites

What strategies can optimize the yield and activity of recombinant Synechocystis sp. cysS?

Optimizing the yield and activity of recombinant cysS requires a systematic approach addressing multiple aspects of protein expression and purification:

• Vector design optimization:

  • Testing different expression vectors with various promoters and fusion tags

  • The pTYB21 vector was successfully used for Synechocystis gene expression

  • Vectors with tightly controlled expression might reduce potential toxicity

  • Codon optimization for the expression host

• Host strain selection:

  • Different E. coli strains offer various advantages:

    • BL21(DE3) strains lack certain proteases, improving protein stability

    • Rosetta strains supply rare tRNAs that might be needed for Synechocystis genes

    • C41/C43 strains are optimized for membrane or toxic protein expression

    • SHuffle strains promote disulfide bond formation in the cytoplasm

• Expression condition optimization:

  • Systematic testing of temperature, media composition, and induction parameters

  • Lower temperatures (16-25°C) often improve folding of heterologous proteins

  • Testing different induction strategies (IPTG concentration, induction time)

  • Specialized media formulations (auto-induction, defined media)

• Co-expression strategies:

  • Co-expression with molecular chaperones (GroEL/ES, DnaK/J) to improve folding

  • Co-expression with tRNA^Cys if the enzyme requires its substrate for proper folding

  • Co-expression with partner proteins if cysS functions in a complex

• Purification optimization:

  • Testing different buffer compositions to maintain enzyme stability

  • Including stabilizing additives (glycerol, reducing agents, specific ions)

  • Optimizing each purification step to minimize activity loss

  • Rapid purification protocols to reduce time-dependent inactivation

Each of these strategies would need to be systematically tested and optimized to determine the conditions that yield the highest amount of active Synechocystis sp. cysS. Documentation of these optimization processes would be valuable for researchers working with similar proteins from cyanobacteria.