Recombinant Acinetobacter baumannii Glycine cleavage system H protein (gcvH)

What is the Glycine cleavage system H protein (gcvH) in Acinetobacter baumannii?

The Glycine cleavage system H protein (gcvH) is a critical component of the glycine cleavage system (GCS) in A. baumannii. It functions as a molecular shuttle within the GCS, which is essential for one-carbon (C1) metabolism . The protein contains a lipoate swinging arm that carries an aminomethyl moiety and transfers it between different proteins in the complex . The protein sequence of recombinant A. baumannii gcvH is: MNHPSELKYARTHEWVKIEGDLVITGITDHAQDELGDLVYVETPEVGSQVTAGEQAGVVESVKTASDIH .

What role does the glycine cleavage system play in bacterial metabolism?

The glycine cleavage system is crucial for one-carbon (C1) metabolism in bacteria, playing a decisive role in many multi-enzyme systems . This system utilizes a lipoate swinging arm containing an aminomethyl moiety attached to protein H, which serves as a molecular shuttle among different proteins in the complex . The protection of this aminomethyl moiety in a cavity of protein H and its release induced by protein T are key processes in the system's function . The GCS is fundamentally important for the utilization of formate and CO₂ for biosynthesis in bacterial cells .

How is recombinant A. baumannii gcvH typically produced for research purposes?

Recombinant A. baumannii gcvH can be produced using various expression systems. According to available product information, the protein can be expressed in E. coli, yeast, baculovirus, or mammalian cell systems . The E. coli-expressed version (product code CSB-EP009335AWO) is commonly used for research purposes . A biotinylated version using Avi-tag technology is also available (CSB-EP009335AWO-B), where BirA catalyzes an amide linkage between biotin and the specific lysine of the AviTag . The final purified protein typically has >85% purity as determined by SDS-PAGE and should be stored at -20°C or -80°C for extended stability .

What is currently known about the structural dynamics of gcvH in the glycine cleavage system?

Molecular dynamics studies of the glycine cleavage system have revealed important structural features of protein H, though not specific to A. baumannii. Research indicates that the protection and release of the lipoate arm of protein H is a complex process involving multiple steps . Based on molecular dynamics simulations of interactions between proteins H and T, four major steps of the release process showing significantly different energy barriers and time scales have been distinguished . Mutations of a key residue, Ser-67 in protein H, have been shown to lead to a bidirectional tuning of the release process, suggesting potential sites for functional modification .

How might gcvH function in A. baumannii pathogenicity, based on findings from related bacterial species?

While the exact role of gcvH in A. baumannii pathogenicity is not directly established in the provided research, evidence from other bacterial species suggests potential pathogenic roles. In Mycoplasma, for example, gcvH has been shown to target the endoplasmic reticulum (ER) to hijack host apoptosis, facilitating bacterial infection . Mechanistically, Mycoplasma gcvH interacts with the ER-resident kinase Brsk2, stabilizing it by blocking its autophagic degradation . This interaction subsequently disturbs unfolded protein response (UPR) signaling, inhibiting the expression of the key apoptotic molecule CHOP and the ER-mediated intrinsic apoptotic pathway . The N-terminal amino acid 31-35 region of Mycoplasma gcvH is necessary for this interaction with Brsk2 . Whether A. baumannii gcvH has similar host-interaction capabilities remains to be determined but represents an intriguing area for investigation.

What comparative analyses can be performed between A. baumannii gcvH and homologous proteins in other bacterial species?

Comparative analyses between A. baumannii gcvH and homologous proteins in other bacterial species can provide valuable insights into its function and evolutionary significance. Researchers could consider:

• Sequence alignment analysis to identify conserved domains and evolutionary relationships

• Structural comparison using molecular modeling techniques to identify functional differences

• Functional assays comparing the biochemical activities of gcvH from different species

• Host-interaction studies to determine if A. baumannii gcvH interacts with host cellular components similar to Mycoplasma gcvH's interaction with the ER

• Comparison with the well-characterized T6SS effectors in A. baumannii to determine if gcvH has any relation to this secretion system that delivers toxic effector proteins to surrounding bacterial cells

What are the optimal conditions for expression and purification of recombinant A. baumannii gcvH?

Based on available information about commercial recombinant A. baumannii gcvH production, researchers should consider the following guidelines for expression and purification:

Expression Systems:

• E. coli: Commonly used and efficient for bacterial protein expression

• Yeast: Alternative system for potential improved folding

• Baculovirus: For higher eukaryotic-like post-translational modifications

• Mammalian cells: For highest authenticity of modifications

Purification Considerations:

• Target purity should be >85% as determined by SDS-PAGE

• Storage at -20°C, with extended storage at -20°C or -80°C recommended

• For specialized applications, consider biotinylated versions using Avi-tag technology, where BirA catalyzes amide linkage between biotin and the specific lysine of the AviTag

The Uniprot accession number A3M4W5 can be referenced for the canonical sequence information .

What methodological approaches can be used to study the structural dynamics of gcvH in vitro?

The study of gcvH structural dynamics can be approached through several sophisticated methodological techniques:

• Molecular Dynamics Simulations: As demonstrated in previous research on the glycine cleavage system, molecular dynamics simulations can effectively reveal the protection and release processes of the lipoate arm . These simulations can identify different energy barriers and time scales involved in conformational changes.

• Site-Directed Mutagenesis: Targeted mutations, such as those performed on Ser-67 in protein H in previous studies, can help identify key residues involved in protein function and modify the dynamics of the release process .

• Structural Analysis Techniques:

  • X-ray crystallography to determine static structure

  • NMR spectroscopy for solution-state dynamics

  • Hydrogen-deuterium exchange mass spectrometry to identify regions involved in conformational changes

• Protein-Protein Interaction Assays:

  • Co-immunoprecipitation to identify binding partners

  • Surface plasmon resonance to measure binding kinetics

  • FRET-based assays to monitor real-time interactions between gcvH and other GCS components

How can researchers investigate potential interactions between A. baumannii gcvH and host cellular components?

To investigate potential interactions between A. baumannii gcvH and host cellular components, researchers could employ approaches similar to those used in studying Mycoplasma gcvH:

• Cell Fractionation Studies: To determine if A. baumannii gcvH localizes to specific cellular compartments such as the endoplasmic reticulum, as observed with Mycoplasma gcvH .

• Protein-Protein Interaction Screening:

  • Yeast two-hybrid screening to identify potential host interaction partners

  • Pull-down assays with recombinant gcvH to isolate interacting host proteins

  • Mass spectrometry analysis of isolated protein complexes

• Domain Mapping: Similar to the identification of the N-terminal amino acid 31-35 region of Mycoplasma gcvH being necessary for Brsk2 interaction , truncation and deletion mutants could identify domains of A. baumannii gcvH involved in host interactions.

• Functional Assays:

  • Apoptosis assays to determine if A. baumannii gcvH affects host cell death pathways

  • ER stress response assays to assess impact on unfolded protein response signaling

  • Autophagy flux assays to examine effects on host autophagic processes

What statistical approaches are most appropriate for analyzing gcvH functional assay data?

When analyzing gcvH functional assay data, researchers should consider:

• Appropriate Controls: Include both positive and negative controls specific to each assay type, especially when comparing wild-type vs. mutant forms of gcvH.

• Statistical Tests:

  • For comparing experimental groups: t-tests (paired or unpaired) for two groups or ANOVA for multiple groups

  • For dose-response relationships: regression analysis

  • For binding kinetics data: non-linear regression for curve fitting

• Replication and Sample Size:

  • Biological replicates (different bacterial cultures) are essential

  • Technical replicates to account for assay variability

  • Power analysis to determine appropriate sample sizes

• Data Visualization:

  • Create consistent graphical representations that highlight key differences

  • Include error bars representing standard deviation or standard error

  • Consider heat maps for complex interaction data

How should researchers address potential strain variability when studying gcvH function in A. baumannii isolates?

A. baumannii demonstrates considerable genetic diversity across different strains, which may extend to gcvH structure and function. Based on observations of variability in Type VI secretion system effectors across A. baumannii strains , researchers should consider:

• Sequence Analysis Across Strains:

  • Compare gcvH sequences from multiple A. baumannii isolates

  • Identify conserved regions versus variable domains

  • Construct phylogenetic trees to understand evolutionary relationships

• Reference Strain Selection:

  • Use well-characterized reference strains such as ATCC 17978 (the source of the recombinant protein in the provided data)

  • Include strains from diverse clinical and environmental sources

• Functional Comparison Framework:

  • Develop standardized assays that can be applied across strains

  • Quantify variation in gcvH expression levels

  • Correlate functional differences with sequence variations

• Contextual Analysis:

  • Consider genomic context and regulatory elements that may differ between strains

  • Assess potential horizontal gene transfer events that might influence gcvH variation

  • Examine correlation between gcvH variants and clinical outcomes or antimicrobial resistance profiles

What approaches can help reconcile contradictory results from different experimental systems studying gcvH?

When faced with contradictory results in gcvH studies across different experimental systems, researchers should:

• Systematic Comparison of Methodologies:

  • Create a detailed table comparing experimental conditions, reagents, and protocols

  • Identify specific variables that differ between studies (pH, temperature, buffer composition)

  • Perform controlled experiments that systematically vary these conditions

• Expression System Considerations:

  • Compare results from different expression systems (E. coli, yeast, baculovirus, mammalian cells)

  • Assess if post-translational modifications affect function

  • Consider effects of tags (His-tag, GST, Avi-tag) on protein function

• Protein Quality Assessment:

  • Verify protein folding and activity through multiple independent methods

  • Consider effects of storage conditions on protein stability and activity

  • Implement rigorous quality control measures for recombinant proteins

• Collaborative Verification:

  • Establish collaborations between labs reporting contradictory results

  • Exchange materials and protocols to directly compare under identical conditions

  • Consider multi-laboratory studies with standardized protocols

What are the most promising therapeutic applications targeting gcvH in A. baumannii infections?

Given the emerging understanding of gcvH function, several therapeutic strategies might be explored:

• Inhibitor Development:

  • Design small molecule inhibitors targeting the gcvH lipoate arm binding cavity

  • Develop peptide-based inhibitors that interfere with gcvH-protein T interactions

  • Screen for natural products that modulate gcvH function

• Host-Interaction Disruption:

  • If A. baumannii gcvH interacts with host components similar to Mycoplasma gcvH's interaction with Brsk2 , develop agents that prevent this interaction

  • Target the N-terminal region that might mediate host interactions, based on findings from homologous proteins

• Vaccine Development:

  • Assess gcvH as a potential vaccine antigen

  • Determine if anti-gcvH antibodies can neutralize its function

• Combination Approaches:

  • Develop strategies that target gcvH alongside other bacterial systems like the Type VI secretion system

  • Explore synergistic effects with conventional antibiotics

What techniques show promise for high-throughput screening of gcvH modulators?

For researchers interested in identifying modulators of gcvH function, several high-throughput screening approaches could be considered:

• Functional Assays:

  • Develop fluorescence-based assays monitoring the protection and release of the lipoate arm

  • Create reporter systems that reflect gcvH activity in bacterial or reconstituted systems

• Binding Assays:

  • Thermal shift assays to identify compounds that stabilize or destabilize gcvH

  • Surface plasmon resonance screening of fragment libraries

  • Differential scanning fluorimetry for rapid compound screening

• Cellular Assays:

  • Phenotypic screens measuring bacterial survival or virulence

  • Host-pathogen interaction models if gcvH is confirmed to interact with host components

• In Silico Approaches:

  • Virtual screening against the gcvH structure

  • Molecular dynamics simulations to identify potential binding sites and predict compound effects