Recombinant Rhodopirellula baltica UPF0176 protein RB8368 (RB8368)

What is known about the basic structural features of the UPF0176 protein RB8368 in Rhodopirellula baltica?

RB8368 belongs to the uncharacterized protein family UPF0176 found in Rhodopirellula baltica. As a member of the planctomycete phylum, R. baltica possesses unique cell compartmentalization and peptidoglycan-less proteinaceous cell walls that inform our understanding of its proteins . While specific structural data on RB8368 is limited, this protein likely reflects some of the novel protein domains and sequence motifs characteristic of this planctomycete. Analysis of the R. baltica genome has revealed several proteins with domains restricted to planctomycetes, suggesting unique evolutionary adaptations . Given its classification as a UPF, RB8368's structure-function relationship remains incompletely characterized, providing an open area for structural biology investigations.

How is RB8368 expression regulated during the Rhodopirellula baltica life cycle?

The expression of RB8368, like many R. baltica proteins, likely varies throughout the organism's growth cycle. Transcriptional profiling of R. baltica has demonstrated that a large number of hypothetical proteins are differentially expressed during the cell cycle . Analysis of gene expression patterns throughout the growth curve shows distinct regulation patterns in different phases. The table below represents the general pattern of gene regulation observed in R. baltica at different growth phases:

This pattern suggests that hypothetical proteins like RB8368 may play critical roles in adaptation to changing environmental conditions throughout the growth cycle . Methodologically, researchers should sample at multiple time points when studying RB8368 expression to capture these potential variations.

What are the optimal conditions for the recombinant expression of RB8368 protein?

The recombinant expression of R. baltica proteins, including RB8368, requires careful optimization of expression systems. Based on research with other R. baltica proteins, the following methodological approach is recommended:

Given that a significant percentage of R. baltica genes encode hypothetical proteins (44-59% of differentially regulated genes) , novel optimization strategies may be necessary for proteins like RB8368.

What are the most effective techniques for detecting post-translational modifications in RB8368?

Post-translational modifications (PTMs) of RB8368 can be investigated using a combination of techniques, with mass spectrometry being particularly powerful. The ubiquitin remnant profiling approach represents an effective strategy for identifying ubiquitination sites in proteins . This methodology involves:

• Sample preparation: Generate tryptic digests of RB8368-containing samples.

• Immunoaffinity enrichment: Use an antibody that selectively binds to the diglycine remnant in peptides generated from tryptic digestion. This approach allows for specific enrichment of ubiquitinated peptides .

• nanoLC-MS/MS analysis: Analyze enriched peptides using high-resolution mass spectrometry. Look for characteristic mass shifts of 114.04 Da corresponding to the Gly-Gly adduct on lysine residues .

• MS data analysis: Identify modified peptides through automated database searching, with manual verification of spectra showing characteristic y- and b-ion series with mass differences of 242.14 Da (lysine + Gly-Gly adduct) .

• Validation: Confirm detected ubiquitination sites through immunoprecipitation with anti-RB8368 antibodies followed by anti-ubiquitin immunoblotting .

For other PTMs, phosphorylation analysis using titanium dioxide enrichment and glycosylation analysis using lectin affinity chromatography would provide complementary information about the modification state of RB8368.

How can we determine the subcellular localization and potential membrane association of RB8368 in R. baltica?

Determining the subcellular localization of RB8368 requires specialized approaches due to the unique cell compartmentalization in R. baltica. Given that planctomycetes have distinct cell plans that may require special membrane transport mechanisms , the following methodological workflow is recommended:

• Bioinformatic prediction: Analyze the RB8368 sequence for potential signal peptides, transmembrane domains, and localization signals. Note that planctomycetes may utilize novel N-terminal export signal peptides distinct from those in other bacteria .

• Fractionation approaches: Perform careful subcellular fractionation of R. baltica cells, separating cytoplasmic, membrane, and potential compartment-specific fractions. Western blot analysis using anti-RB8368 antibodies can identify which fraction contains the protein.

• Fluorescence microscopy: Create fluorescent protein fusions (GFP-RB8368) for expression in R. baltica and visualize localization patterns in living cells. Compare localization patterns throughout the growth cycle, as localization may change in response to environmental conditions .

• Immunogold electron microscopy: For highest resolution localization, perform immunogold labeling with anti-RB8368 antibodies followed by transmission electron microscopy. This approach is particularly valuable for proteins associated with the unique membrane systems of planctomycetes.

• Membrane association analysis: Treat membrane fractions with various reagents (high salt, pH extremes, mild detergents) to determine the nature of membrane association if present.

It's worth noting that ubiquitination analysis of other proteins has shown that subcellular distribution of ubiquitinated proteins generally indicates primarily cytosolic localization, with some nuclear and mitochondrial proteins also being ubiquitinated .

What are the potential interaction partners of RB8368 and how can they be experimentally validated?

Identifying interaction partners of RB8368 requires a multi-faceted approach:

• Affinity purification-mass spectrometry (AP-MS): Express His-tagged or FLAG-tagged RB8368 in R. baltica or heterologous systems, perform affinity purification under native conditions, and identify co-purifying proteins by mass spectrometry.

• Yeast two-hybrid screening: Use RB8368 as bait to screen for interacting proteins from an R. baltica cDNA library. This approach can identify direct protein-protein interactions.

• Proximity labeling: Express RB8368 fused to a promiscuous biotin ligase (BioID or TurboID) to biotinylate proximal proteins in vivo, followed by streptavidin purification and mass spectrometry.

• Co-immunoprecipitation validation: Validate key interactions by co-immunoprecipitation experiments with antibodies against RB8368 and suspected interaction partners.

• Functional validation: Assess the impact of RB8368 knockout or overexpression on the function or localization of identified interaction partners.

Given the high percentage of hypothetical proteins in R. baltica (56-58% of differentially regulated genes) , it's likely that some interaction partners will also be uncharacterized proteins, creating interesting opportunities for network analysis of novel protein functions.

What experimental approaches can determine if RB8368 undergoes ubiquitination and how this affects its function?

To investigate potential ubiquitination of RB8368, researchers should employ a systematic approach:

• Ubiquitin remnant profiling: Apply the anti-diglycine antibody immunoaffinity approach coupled with nanoLC-MS/MS to identify potential ubiquitination sites in RB8368 . This technique has successfully identified hundreds of ubiquitination sites in mammalian proteins.

• Site-directed mutagenesis: Once potential ubiquitination sites are identified, create lysine-to-arginine mutants at these positions and assess functional consequences.

• SILAC-based quantitative proteomics: Use SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) approaches to quantify changes in RB8368 ubiquitination under different experimental conditions, such as different growth phases or stress conditions .

• Ubiquitination assays: Perform in vitro ubiquitination assays with purified RB8368, E1, E2, and candidate E3 ligases to confirm direct ubiquitination.

• Functional consequences: Assess how ubiquitination affects RB8368 stability, localization, or activity using ubiquitination-deficient mutants compared to wild-type protein.

The table below outlines potential experimental conditions for analyzing dynamic ubiquitination of RB8368:

This approach parallels successful studies of dynamic ubiquitination in other proteins like PCNA and tubulin α-1A .

How does RB8368 expression correlate with the unique physiological adaptations of R. baltica across different growth conditions?

Understanding the relationship between RB8368 expression and R. baltica's physiological adaptations requires integration of transcriptomic and physiological data:

• Growth phase analysis: Monitor RB8368 expression across the growth curve using RT-qPCR or proteomics. R. baltica shows significant transcriptional changes across growth phases, particularly in the transition to stationary phase when 12% of genes are differentially regulated .

• Stress response profiling: Expose R. baltica to various stressors (nutrient limitation, salinity changes, oxidative stress) and assess RB8368 expression changes. Many R. baltica proteins show differential regulation under stress conditions .

• Physiological parameter correlation: Correlate RB8368 expression levels with specific physiological changes, such as cell morphology alterations, adhesion capability, or metabolism shifts.

• Knockout/knockdown studies: Generate RB8368-deficient strains and characterize phenotypic consequences across different growth conditions.

Research on R. baltica has shown that transition from exponential to stationary phase triggers significant metabolic adaptations, including induction of stress-response proteins like glutathione peroxidase (RB2244), thioredoxin (RB12160), and universal stress protein (uspE, RB4742) . If RB8368 shows similar expression patterns, it may play a role in long-term survival under unfavorable conditions.

Of particular interest would be determining whether RB8368 expression correlates with the formation of rosettes and holdfast substances, which are characteristic of R. baltica's attachment phase and influenced by growth conditions .

How does RB8368 compare to other UPF0176 family proteins across different bacterial species?

A comparative analysis of RB8368 with other UPF0176 family proteins would provide evolutionary insights using the following methodological approaches:

• Sequence analysis: Perform multiple sequence alignment of UPF0176 family proteins from diverse bacterial species to identify conserved residues and planctomycete-specific variations.

• Phylogenetic analysis: Construct phylogenetic trees to understand the evolutionary relationships between RB8368 and homologs from other bacteria, potentially revealing whether UPF0176 proteins in planctomycetes underwent lineage-specific adaptations.

• Domain architecture comparison: Analyze if RB8368 contains any of the novel domains identified in R. baltica, such as the GEFGR protein family or ASPIC C-terminal domain family .

• Structural prediction and comparison: Generate structural models of RB8368 and homologs to identify conserved structural features despite sequence divergence.

The unique cell biology of planctomycetes suggests that proteins like RB8368 may have undergone specific adaptations to support the distinctive cellular compartmentalization of these organisms . A comparative approach would help determine whether RB8368 represents a planctomycete-specific adaptation or maintains conserved functions across bacterial phyla.

What methodological approaches can elucidate the potential role of RB8368 in the unique cell compartmentalization of R. baltica?

R. baltica's distinctive cell compartmentalization may involve specialized proteins like RB8368. To investigate this possibility:

• Localization studies: Perform high-resolution immunolocalization to determine if RB8368 associates with specific membrane compartments or cellular structures unique to planctomycetes.

• Protein-lipid interaction analysis: Assess RB8368 binding to different lipids using lipid overlay assays or liposome flotation assays to determine membrane preference.

• Structural analysis: Determine the three-dimensional structure of RB8368 using X-ray crystallography or cryo-EM to identify potential membrane-interacting regions.

• Domain function analysis: Create domain deletion constructs to identify regions essential for proper localization or function.

• Comparative expression analysis: Compare RB8368 expression with genes known to be involved in maintaining R. baltica's unique cell organization.

The search results indicate that many R. baltica-specific protein domains are associated with secretion and cell-surface functions , suggesting proteins like RB8368 could be involved in establishing or maintaining the unique cell compartmentalization. The discovery that one novel motif in R. baltica likely represents a novel N-terminal export signal peptide highlights the specialized membrane transport mechanisms that might involve proteins like RB8368.