Dengue Envelope-2 45kDa

What is Dengue Envelope-2 45kDa protein and what is its role in viral infection?

Dengue Envelope-2 (E-2) 45kDa is a recombinant surface glycoprotein from Dengue Virus Subtype 2, one of four serotypes in the genus Flavivirus, family Flaviviridae. The protein plays crucial roles in viral attachment to host cells, membrane fusion, and entry into target cells. The protein typically contains amino acids 2-395 or 43-413 (depending on the expression system) with a molecular weight of approximately 45.3 kDa .

The E protein functions through several critical processes: regulating fusion-loop exposure through shielding, tethering, and triggered release; enabling hinge movements between domains during structural transformations; and driving membrane fusion through interactions with the stem region . These functions make the E protein essential for viral infectivity and a primary target for neutralizing antibodies.

What structural domains compose Dengue Envelope-2 and how do they function together?

Dengue Envelope-2 consists of three distinct domains (DI, DII, and DIII) connected by flexible hinge regions that enable conformational changes during viral entry:

• Domain I (DI): Central structural domain that serves as an organizational hub

• Domain II (DII): Contains the fusion loop critical for membrane fusion

• Domain III (DIII): Primarily involved in receptor binding and a major target for neutralizing antibodies

The flexible hinge regions connecting these domains are particularly important as they contain "underpacked regions" that facilitate the necessary conformational changes during infection . These hinges are not optimized for maintaining a stable dimer conformation, but rather for enabling the protein to transition between multiple conformational states during the viral life cycle . This strategic flexibility is essential for the protein's function in membrane fusion.

How are different recombinant forms of Dengue Envelope-2 45kDa produced?

Recombinant Dengue Envelope-2 45kDa protein can be produced in multiple expression systems with different characteristics:

Each expression system offers distinct advantages. E. coli production typically yields higher amounts of protein and is more cost-effective, while insect cell expression may produce protein with more native-like post-translational modifications . Recent research has identified mutations that can significantly enhance production yield (>50-fold) while inducing dimerization at low concentrations, offering improved options for researchers requiring larger quantities of functional protein .

What key residues in Dengue Envelope-2 mediate membrane fusion?

Comprehensive structure-function analysis covering all 390 residues of the dengue virus E protein ectodomain has identified specific residues critical for virus infectivity that do not affect protein expression, folding, virion assembly, or budding . These functionally critical residues include:

• Fusion-loop regulators: Residues that shield, tether, and trigger the release of the fusion loop

• Hinge residues: Amino acids enabling movement between domain interfaces during structural transformations

• Membrane fusion drivers: Residues forming "late-stage zipper contacts with stem" that drive membrane fusion

Histidine residues play an especially important role as "pH-sensing" elements that mediate the E protein conformational change from prefusion dimer to postfusion trimer . These residues respond to the acidic environment of the endosome, triggering the conformational changes necessary for membrane fusion. This functional understanding provides specific targets for therapeutic development aimed at disrupting viral entry.

How do pH changes affect the conformational states of Dengue Envelope-2?

The Dengue Envelope-2 protein undergoes pH-dependent conformational changes that are essential for virus fusion with host cell membranes. These changes are mediated primarily through "histidine-cation interactions" that act as pH-sensing elements . These histidine residues are not optimized for maintaining dimer stability but rather for responding to environmental pH changes - representing an evolutionary trade-off between stability and functional responsiveness.

At neutral pH, the E protein exists as a dimer on the virus surface. When exposed to the acidic environment of the endosome (lower pH), protonation of key histidine residues disrupts specific interactions maintaining the dimeric conformation. This triggers a cascade of conformational changes involving the flexible hinge regions between domains, ultimately resulting in exposure of the fusion loop and formation of the postfusion trimer that drives membrane fusion . Understanding this pH-dependent mechanism has significant implications for antiviral development targeting viral entry.

What approaches have been used to stabilize Dengue Envelope-2 for research applications?

Researchers have employed several strategies to enhance Dengue Envelope-2 stability for various applications:

• Designed mutations: Through comprehensive molecular modeling (>7,000 simulations per structure), researchers identified mutations that "induce dimerization at low concentrations (<100 pM) and enhance production yield by more than 50-fold" .

• Cysteine engineering: Specific variants including "Cm1 (A259C) and Cm2 (L107C and A313C)" were designed to stabilize protein structure through disulfide bond formation .

• Structure-guided design: Design protocols incorporated "a design sphere in which all residues within 7 Å of a seed residue were allowed to mutate to any amino acid except cysteine," while preserving residues known to interact with neutralizing antibodies .

• Buffer optimization: Addition of stabilizers like D-trehalose and protease inhibitors (Pepstatin A, Leupeptin, Aprotinin) helps maintain protein integrity .

For long-term storage, researchers recommend keeping the protein below -18°C to -20°C and adding carrier proteins (0.1% HSA or BSA) . Multiple freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity .

How can researchers distinguish between monomeric and dimeric forms of Dengue Envelope-2?

Distinguishing between monomeric and dimeric forms of Dengue Envelope-2 is essential for many research applications. Several approaches can be employed:

• Engineered controls: Researchers have designed variants that preferentially form either dimers (Cm1, Cm2) or monomers (Mnmer1, Mnmer2) that serve as experimental controls .

• Concentration and temperature manipulation: Wild-type protein dimerization is concentration and temperature-dependent, with certain engineered variants remaining monomeric "even at higher concentrations and low temperature" .

• Antibody-based detection: Researchers have identified antibodies "directed to share epitopes on monomers and dimers or to dimer-specific epitopes" . Depletion experiments where "monomer depletion will selectively remove the Abs low and promote the binding of dimer-specific Abs high" can further distinguish conformational states .

• Binding kinetics analysis: Mathematical binding models simulating "lower-affinity (K d = 10 nM) and higher-affinity (K d = 0.1 nM) antibody binding" help interpret complex binding patterns in antibody assays .

These approaches enable researchers to work with defined conformational states, which is critical for structure-function studies and vaccine development targeting specific conformations.

What methods are effective for studying Dengue Envelope-2 interactions with host receptors?

The search results reveal several effective approaches for studying E protein-receptor interactions:

• Protein-protein interaction assays: DENV2 pretreated with trypsin was shown to interact with a 31 kDa protein (AAEL011180) in mosquito midgut, identified through protein mass spectrometry .

• Surface plasmon resonance (SPR): This technique demonstrated that "recombinant DENV2 surface E glycoprotein bound to recombinant AAEL011180 with high affinity (38.2 nM)" .

• Genetic validation approaches:

  • Gene knockout: Studies disrupting the gene encoding the putative receptor (EPrRec) confirmed its role, though complete knockout wasn't possible as "the gene is essential in Ae. aegypti"

  • RNA interference: "Reducing EPrRec mRNA expression in the midgut of ΔEPrRec +/− females by systemic dsRNA injection significantly reduced the prevalence of DENV2 infection"

• Immobilized receptor binding: Confirmation that "virus also bound to immobilized recombinant purified receptor" provided additional evidence of direct interaction .

These methodologies provide a robust framework for identifying and validating virus-receptor interactions, which is critical for understanding transmission mechanisms and developing intervention strategies.

What are the critical quality control parameters for Dengue Envelope-2 45kDa preparations?

Several key parameters should be assessed to ensure the quality of Dengue Envelope-2 45kDa preparations:

• Purity assessment: Both commercial sources report >95% purity determined by SDS-PAGE (10-12.5%) with Coomassie staining . This represents the minimum acceptable purity standard for research applications.

• Structural integrity: While not explicitly detailed in the search results, proper folding and conformational state of the protein are critical quality parameters that would typically be assessed through:

  • Circular dichroism spectroscopy

  • Intrinsic fluorescence measurements

  • Thermal stability assays

• Functional activity: Activity can be assessed through:

  • Receptor binding assays

  • Antibody recognition

  • Liposome fusion assays (for fusion-competent preparations)

• Storage stability: Stability should be monitored under recommended storage conditions:

  • 4°C (short-term, 1-4 weeks)

  • -18°C to -20°C (long-term)

  • With carrier proteins (0.1% HSA or BSA) for extended storage

• Endotoxin levels: For preparations intended for immunological studies, endotoxin levels should be monitored and minimized to prevent non-specific immune activation.

These quality control parameters ensure that research results obtained with the protein are reliable and reproducible.

How can Dengue Envelope-2 structure-function analysis inform vaccine development?

The detailed structural and functional characterization of Dengue Envelope-2 protein provides critical insights for rational vaccine design:

• Identification of conserved functional epitopes: Comprehensive mutagenesis studies have identified "residues that are critical for virus infectivity" that represent potential targets for vaccine-induced immunity . These functionally constrained regions may be less prone to escape mutations.

• Conformationally stabilized immunogens: Engineered stable E protein dimers (SC.14 and SC.10) "elicit DENV2 E dimer-specific antibodies in mice," suggesting that stabilized conformations might serve as improved vaccine antigens by presenting conformation-specific epitopes .

• Fusion mechanism targeting: The "cohesive mechanistic model" describing "dynamic processes by which specific residue interactions within Envelope mediate infectivity" provides specific functional targets for vaccine design . Antibodies that block these critical steps could effectively neutralize the virus.

• Cross-serotype considerations: Given that "one might have dengue fever infected by the different serotype virus after the primary infection," vaccines must address all four dengue serotypes to prevent antibody-dependent enhancement . Structure-function analysis helps identify conserved epitopes across serotypes.

This structure-based approach to vaccine design offers the potential to overcome challenges associated with traditional vaccine approaches by focusing immune responses on functionally critical, conserved epitopes rather than variable regions.

What novel screening approaches can identify inhibitors targeting Dengue Envelope-2 functions?

While the search results don't directly address screening approaches for inhibitors, several methodologies can be inferred based on the structural and functional information provided:

• Structure-guided virtual screening: The detailed atomic-level understanding of E protein function provides a foundation for in silico screening campaigns targeting specific functional sites, such as:

  • The fusion loop and its regulatory residues

  • Hinge regions critical for conformational changes

  • Dimer interface stabilizing interactions

  • pH-sensing histidine residues

• Mammalian surface display: The search results mention "epitope presentation screen of DENV2 sE Rosetta variants using mammalian surface display" . This approach could be adapted to screen for small molecules or peptides that bind to specific conformations or functional regions of the E protein.

• Conformation-specific binding assays: Given the engineering of stable monomeric and dimeric variants, high-throughput assays could be developed to identify compounds that selectively stabilize pre-fusion conformations or prevent pH-induced conformational changes.

• Fusion inhibition assays: In vitro liposome fusion assays could be employed to screen for compounds that directly inhibit the membrane fusion function of the E protein.

These approaches, guided by the detailed structural and functional insights available for the E protein, offer promising avenues for the discovery of novel antivirals targeting dengue virus entry.

How does Dengue Envelope-2 compare to envelope proteins from other flaviviruses?

The Dengue Envelope-2 protein shares structural and functional features with envelope proteins from other flaviviruses, with important implications for comparative research:

• Conserved fusion mechanism: The atomic-level functional model for DENV2 E protein "may also apply to related class II viral fusion proteins" . This suggests that the fundamental fusion mechanism involving pH-sensing, conformational changes, and membrane fusion is conserved across flaviviruses.

• Receptor interactions: The identified "DENV2 E protein receptor, EPrRec" in mosquitoes is "a highly conserved protein and has orthologs in culicine and anopheline mosquitoes" . This suggests potential conservation of vector-virus interactions across different flavivirus-mosquito systems.

• Domain organization: The three-domain architecture (DI, DII, DIII) connected by flexible hinge regions appears to be conserved across flavivirus envelope proteins, though the specific residues in these domains may vary.

• Serotype variation: Even within dengue viruses, there is "difficulty for manufacturers to have the dengue antigens with a whole coverage for Dengue IgG & IgM recognition for all 4 serotype infections" . This highlights the antigenic variability that exists even among closely related viruses.

This comparative understanding can guide development of broad-spectrum interventions targeting conserved mechanisms while also informing serotype-specific approaches where necessary. Research methodologies developed for DENV2 E protein can potentially be applied to other flaviviruses of public health significance, such as Zika, West Nile, and yellow fever viruses.

What are the optimal storage conditions for maintaining Dengue Envelope-2 45kDa stability?

Proper storage conditions are essential for maintaining the structural integrity and functionality of Dengue Envelope-2 45kDa. Based on the commercial sources, the following storage guidelines are recommended:

The ProspecBio formulation includes "1xD-PBS, pH7.4, 1μg/mL Pepstatin A, 0.099% Thimerosal, 1μg/mL Leupeptin, 1μg/mL Aprotinin, & 2.5% D-trehalose" to enhance stability . The Abbexa preparation recommends storage in "25 mM Tris base and 10 mM K₂CO₃" .

Both sources emphasize avoiding repeated freeze-thaw cycles as these can lead to protein denaturation, aggregation, and loss of functional activity . For critical applications, researchers should validate the structural integrity and activity of stored protein before use, particularly after extended storage periods.

What are common troubleshooting approaches for experiments involving Dengue Envelope-2?

While the search results don't directly address troubleshooting, several common challenges and solutions can be inferred:

• Protein aggregation:

  • Ensure proper storage conditions are maintained

  • Add carrier proteins (0.1% HSA or BSA) for stability

  • Include stabilizers like D-trehalose in buffer formulations

  • Centrifuge protein solution before use to remove aggregates

• Inconsistent antibody binding:

  • Consider conformational heterogeneity (monomer vs. dimer)

  • Use engineered variants with stabilized conformations

  • Be aware that some antibodies may recognize shared epitopes while others are conformation-specific

• Low protein yield:

  • Consider using engineered variants with enhanced expression (>50-fold improvements reported)

  • Optimize expression conditions (temperature, induction timing)

  • Explore alternative expression systems (E. coli vs. insect cells)

• pH-dependent conformational changes interfering with experiments:

  • Carefully control buffer pH in experimental setups

  • Consider using stabilized variants resistant to pH-induced conformational changes

  • Include appropriate controls to account for pH effects

• Receptor binding variability:

  • Ensure recombinant protein maintains native-like conformation

  • Control for batch-to-batch variation in binding studies

  • Include positive controls with validated binding activity

These troubleshooting approaches address common challenges encountered when working with conformationally dynamic viral envelope proteins like Dengue Envelope-2.

What ethical and biosafety considerations apply to research with Dengue Envelope-2 45kDa?

While the recombinant Dengue Envelope-2 45kDa protein itself does not pose infectious risks, several important considerations apply:

• Research purpose limitations: Both commercial sources explicitly state that their products are "FOR LABORATORY RESEARCH USE ONLY" and specifically not for "diagnostic, therapeutic or cosmetic procedures" or "human or animal consumption" .

• Material source disclosure: When using these materials in research publications, researchers should disclose the source, catalog number, and production system to ensure reproducibility.

• Risk assessment: While the recombinant protein itself is non-infectious, research involving functional assays (such as cell binding or fusion assays) should be assessed for potential risks.

• Comparative studies with infectious virus: Any parallel studies comparing recombinant protein function to live dengue virus would require appropriate biosafety level containment (typically BSL-2 for dengue virus work).

• Development of therapeutics or diagnostics: If research findings lead to potential diagnostic or therapeutic applications, additional regulatory considerations would apply before clinical translation.

Researchers should consult their institutional biosafety committees when designing studies involving viral proteins, even non-infectious recombinant ones, to ensure compliance with all applicable guidelines and regulations.