Recombinant Vibrio vulnificus UPF0299 membrane protein VV1471 (VV1471)

What is VV1471 and what are its basic structural characteristics?

VV1471 is a UPF0299 family membrane protein found in the opportunistic pathogen Vibrio vulnificus. The full-length protein consists of 123 amino acids with the sequence: MKFSLKDLFGLVVSFGLIFLALTIGSGIQHWTGTSVPGSVIGMLVLFVSMAIGLVKVEWVKPGASLLIRYMILLFVPISVGLMEHFDMLIANALPIIASAIGGSLIVLVSLGWLLQRILGKEA . As a membrane protein, it contains hydrophobic regions that facilitate its integration into the bacterial cell membrane. The protein is identified in UniProt with ID Q7MLF6, and its gene designation is VV1471 .

What expression systems are most effective for producing recombinant VV1471?

E. coli expression systems have been successfully employed for the production of recombinant VV1471. For optimal expression, the protein is typically fused with an N-terminal His-tag to facilitate purification using affinity chromatography . When designing expression protocols, researchers should consider that membrane proteins often require careful optimization of induction conditions, detergent selection for solubilization, and temperature management during expression to prevent aggregation or misfolding. The established protocol yields recombinant VV1471 with greater than 90% purity as determined by SDS-PAGE analysis .

How should recombinant VV1471 be stored and reconstituted for experimental use?

For long-term storage, recombinant VV1471 should be maintained at -20°C to -80°C in aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity . The lyophilized protein is typically stored in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For reconstitution, it is recommended to:

• Briefly centrifuge the vial prior to opening to collect contents at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard) for storage at -20°C/-80°C

Working aliquots may be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided to maintain protein stability and activity .

What are the recommended approaches for studying VV1471's membrane localization and topology?

To effectively study VV1471's membrane localization and topology, researchers should employ multiple complementary techniques. Computational prediction tools can initially identify transmembrane domains based on the amino acid sequence. For experimental validation, techniques such as:

• Protease protection assays - to determine which protein regions are accessible

• Site-directed mutagenesis followed by functional assays

• Fluorescent protein fusion constructs for visualization

• Cryo-electron microscopy (cryo-EM) - particularly valuable as it has been successfully used to study membrane structures in Vibrio vulnificus

When designing cryo-EM experiments for membrane protein visualization, researchers should optimize sample preparation conditions and consider using a similar approach to that used for visualizing outer membrane vesicles in Vibrio vulnificus, which revealed distinct organizational patterns dependent on the bacterial strain characteristics .

How can researchers design experiments to investigate potential interactions between VV1471 and other bacterial membrane components?

When investigating VV1471's interactions with other membrane components, researchers should implement a systematic experimental design approach:

• Formulate a specific, testable hypothesis about the interaction

• Clearly define independent variables (e.g., presence/absence of potential interaction partners) and dependent variables (e.g., binding affinity, functional changes)

• Design experimental treatments that manipulate the independent variable

• Plan appropriate control experiments

• Select measurement techniques suitable for membrane protein interactions

Appropriate techniques include co-immunoprecipitation, bacterial two-hybrid systems, FRET-based assays, and crosslinking approaches. When studying potential interactions with capsular components, researchers should consider examining both wild-type and unencapsulated mutant strains, as capsular polysaccharides have been shown to influence membrane organization in Vibrio vulnificus .

How might VV1471 contribute to Vibrio vulnificus pathogenicity and virulence mechanisms?

While direct evidence of VV1471's role in virulence is limited in the available literature, researchers can design investigations based on our understanding of Vibrio vulnificus pathogenicity mechanisms. Vibrio vulnificus is an opportunistic pathogen that can cause acute gastroenteritis, invasive septicemia, tissue necrosis, and potentially death . Key virulence factors include capsular polysaccharide (CPS), lipopolysaccharide, flagellum, pili, and outer membrane vesicles (OMVs) .

As a membrane protein, VV1471 could potentially contribute to pathogenicity through:

• Involvement in membrane organization or stability

• Participation in secretion systems for virulence factors

• Role in antimicrobial resistance mechanisms

• Contribution to outer membrane vesicle formation or regulation

Experimental approaches to investigate these possibilities would include generating knockout mutants and comparing their virulence to wild-type strains in appropriate infection models, as well as examining the protein's potential association with known virulence structures like OMVs.

What is the relationship between VV1471 and outer membrane vesicle (OMV) formation in Vibrio vulnificus?

Research has established that Vibrio vulnificus produces outer membrane vesicles (OMVs) that play a role in virulence . While specific information on VV1471's direct involvement in OMV formation is not explicitly provided in the available literature, researchers investigating this relationship should consider:

• Comparing OMV production between wild-type and VV1471 knockout mutants

• Examining VV1471 protein localization in relation to OMV formation sites

• Analyzing OMV composition for the presence of VV1471

The unique organization pattern of OMVs in Vibrio vulnificus, particularly the regular concentric rings observed in wild-type cells (spacing ~200 nm between rings) versus the irregular spacing in unencapsulated mutants, suggests that capsular polysaccharides regulate aspects of OMV production . This relationship could inform studies of membrane proteins like VV1471 that might participate in these processes.

Table 1: Comparison of OMV characteristics across Vibrio vulnificus strains based on cryo-EM analysis

How can researchers design selective inhibitors targeting VV1471 for potential therapeutic applications?

Designing selective inhibitors against VV1471 would require a methodical research approach:

• Structural characterization: Determine the three-dimensional structure of VV1471 using X-ray crystallography, NMR, or cryo-EM to identify potential binding pockets

• In silico screening: Use computational methods to screen for compounds that might bind to identified pockets

• Binding assays: Develop assays to measure direct binding of candidate compounds to purified VV1471

• Functional validation: Test whether compounds that bind VV1471 inhibit its function

• Specificity testing: Evaluate compounds against homologous proteins from host organisms to ensure selectivity

Given VV1471's membrane localization, researchers should account for membrane penetration capabilities of potential inhibitors and consider using liposome-based delivery systems for in vitro testing. The amino acid sequence provided in the literature can serve as a starting point for structural prediction and identification of functionally important residues that might be targeted.

What approaches can be used to investigate post-translational modifications of VV1471 and their functional significance?

Investigating post-translational modifications (PTMs) of VV1471 requires a multi-faceted experimental approach:

• Mass spectrometry (MS) analysis of purified VV1471 to identify potential PTMs

• Site-directed mutagenesis of identified or predicted modification sites

• Functional comparison of wild-type and mutant proteins

• Temporal analysis of modifications during different growth phases

• Examination of modification enzymes present in Vibrio vulnificus

Researchers should pay particular attention to modifications common in bacterial membrane proteins, such as lipidation, glycosylation, and phosphorylation. When designing such experiments, consider that Vibrio vulnificus shows differences in protein expression and structure during different growth phases, as evidenced by the observation that OMV production is limited and irregular during stationary growth phase compared to log-phase growth .

How does VV1471 compare to homologous proteins in other Vibrio species and bacterial pathogens?

A comprehensive comparative analysis of VV1471 with homologous proteins in other bacterial species would include:

• Sequence alignment analysis to identify conserved domains and variable regions

• Phylogenetic tree construction to understand evolutionary relationships

• Structural comparison of homologs with available 3D structures

• Functional comparison through complementation studies

This evolutionary perspective can provide insights into the protein's core functions (represented by highly conserved regions) versus adaptations specific to Vibrio vulnificus (represented by divergent regions). Such analysis should consider both closely related Vibrio species and more distant bacterial pathogens with UPF0299 family proteins.

What experimental approaches would best determine if VV1471 undergoes structural changes under different environmental conditions relevant to Vibrio vulnificus pathogenesis?

To investigate VV1471's potential structural adaptations to environmental conditions, researchers should design experiments that:

• Express and purify VV1471 under conditions mimicking different host environments (varying temperature, pH, salt concentration)

• Employ circular dichroism spectroscopy to detect secondary structure changes

• Use fluorescence spectroscopy to monitor tertiary structure alterations

• Apply hydrogen-deuterium exchange mass spectrometry to identify regions with altered solvent accessibility

• Perform functional assays under these varying conditions to correlate structural changes with functional outcomes

This approach aligns with established experimental design principles by clearly defining variables, controlling for confounding factors, and systematically testing how environmental conditions (independent variables) affect protein structure and function (dependent variables).