Recombinant Uperoleia mjobergii Uperin-3.5
Product Specs
Target Background
Q&A
What is Uperin-3.5 and what is its biological origin?
Uperin-3.5 is an antimicrobial peptide isolated from the Australian toadlet Uperoleia mjobergii. It belongs to a diverse group of peptides that evolved across the tree of life to combat microbial challenges . This peptide has gained significant attention for its ability to form amyloid-like fibrils that correlate with its antimicrobial function, challenging the traditional view that amyloids are exclusively pathological structures .
What are the unique structural features of Uperin-3.5?
The most remarkable feature of Uperin-3.5 is its structural polymorphism. It can form two distinct types of amyloid fibrils:
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Cross-α fibrils: In the presence of bacterial membrane lipids, Uperin-3.5 forms fibrils with a helical architecture, where antiparallel α-helices stack into sheets that mate via a hydrophobic core .
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Cross-β fibrils: In the absence of lipids, Uperin-3.5 transitions to the more typical amyloid cross-β architecture, forming fibrils with a 3-blade symmetrical propeller arrangement consisting of nine peptides per fibril layer with tight β-sheet interfaces .
This conformational flexibility appears to be functionally relevant, serving as a potential regulatory mechanism for its antimicrobial activity .
How does the crystal structure of Uperin-3.5 compare to other antimicrobial peptides?
| Feature | Uperin-3.5 | PSMα3 |
|---|---|---|
| Helix orientation | Antiparallel | Parallel |
| Inter-sheet distance | Shorter | Longer |
| Inter-helix distance | Shorter | Longer |
| Shape complementarity | Smaller | Larger |
| Sheet arrangement | Staggered orientation | Not staggered |
These differences demonstrate that the cross-α amyloid architecture can be achieved through different molecular arrangements, highlighting the structural versatility of amyloid fibrils .
What techniques are optimal for producing recombinant Uperin-3.5?
While the search results don't provide specific recombinant production protocols, researchers working with antimicrobial peptides typically employ bacterial expression systems with careful consideration of the peptide's characteristics. When producing Uperin-3.5, researchers should account for:
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Expression system selection that balances yield against potential toxicity to the host
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Purification strategies that address the peptide's propensity to form fibrils
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Buffer conditions that maintain stability while preventing premature aggregation
The published studies utilized recombinant Uperin-3.5, confirming that successful expression and purification are achievable .
How can researchers distinguish between cross-α and cross-β fibril forms experimentally?
Multiple complementary techniques are necessary to definitively characterize the different fibril forms:
| Technique | Cross-α Fibrils | Cross-β Fibrils |
|---|---|---|
| Solid-state circular dichroism (ssCD) | Minima at 208 nm and 222 nm (222 nm deeper in fibrils) | Minimum at 218 nm |
| Transmission electron microscopy | Fibril morphology varies with conditions | Fibril morphology varies with conditions |
| Heat stability | Less thermostable | More thermostable |
| X-ray diffraction | Pattern characteristic of α-helical arrangement | Pattern characteristic of β-sheet arrangement |
The ssCD spectrum is particularly informative, as Uperin-3.5 in α-helical conformation shows a deeper 222 nm minimum compared to the 208 nm minimum in fibrillar form, while the opposite trend is observed in solution .
What environmental conditions influence Uperin-3.5 structure and function?
The structure and function of Uperin-3.5 are highly dependent on environmental factors:
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Lipid presence: When incubated with bacterial membrane lipids (DOPE:DOPG SUVs), Uperin-3.5 forms α-helical structures and cross-α fibrils, which correlates with enhanced antimicrobial activity .
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Temperature: Heat shock treatment causes Uperin-3.5 fibrils to transition from predominantly α-helical to β-rich conformation. This structural transition reduces antimicrobial activity, with the minimum inhibitory concentration (MIC) increasing from 4 μM to 16 μM after heat treatment .
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Bacterial cell interaction: Uperin-3.5 forms massive fibrils around and on bacterial cells, leading to membrane damage and bacterial death. This indicates a bi-directional relationship where bacterial lipids induce cross-α fibrillation, and the resulting fibrils exert antimicrobial activity .
What is the proposed mechanism for Uperin-3.5's antimicrobial activity?
Based on the research findings, the antimicrobial mechanism of Uperin-3.5 appears to involve:
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Initial interaction with bacterial membrane lipids
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Transition to α-helical conformation and formation of cross-α fibrils
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Accumulation of fibrils on the bacterial cell surface
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Membrane disruption, leading to cell permeation and bacterial death
Evidence supporting this mechanism includes the observation of massive fibril formation around M. luteus cells, membrane dents visible by electron microscopy, and propidium iodide uptake indicating cell death following exposure to Uperin-3.5 .
The secondary structure switch between cross-α and cross-β forms appears to regulate activity, with the cross-α form being the active antibacterial agent and the cross-β form potentially serving as a storage form with reduced activity .
How can mutations be designed to probe structure-function relationships?
Strategic mutations could target key structural elements of Uperin-3.5:
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Inter-helical interaction sites: Mutations of residues involved in electrostatic interactions (Asp4, Lys14) and hydrogen bonding (Arg7) between adjacent helices in the cross-α fibril could affect fibril stability and antimicrobial activity .
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Hydrophobic core residues: Altering amino acids at the hydrophobic interface between mated sheets could probe the importance of this interface for fibril formation and stability.
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Amphipathicity modifications: Previous studies with Uperin-3.5 mutants have shown that high α-helical content and lower net charge contribute to higher aggregation rates, suggesting targets for mutation to modulate the peptide's behavior .
What is known about the relationship between Uperin-3.5 fibril structure and thermostability?
The research reveals interesting connections between fibril structure and thermostability:
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Cross-β fibrils of Uperin-3.5 demonstrate higher thermostability compared to cross-α fibrils .
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Heat shock treatment causes a transition from α-helical to β-rich conformation in Uperin-3.5 fibrils, which correlates with increased thermostability .
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This pattern extends beyond Uperin-3.5: S. aureus PSMα1 fibrils with cross-β configuration are thermostable, while PSMα3 cross-α fibrils dissolve after heat shock treatment .
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The presence of lipids and heat have opposite effects on secondary structure, suggesting a complex interplay between environmental factors in determining fibril properties .
How does Uperin-3.5 contribute to our understanding of amyloid-antimicrobial relationships?
Uperin-3.5 provides significant insights into the evolutionary and functional connections between amyloids and antimicrobial peptides:
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Cross-kingdom structural conservation: The finding that both amphibian Uperin-3.5 and bacterial PSMα3 form cross-α amyloid fibrils suggests this structural architecture exists across kingdoms of life and may have played functional roles in early evolution .
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Support for the amyloid-antimicrobial link hypothesis: These findings support the hypothesis that antimicrobial properties and amyloid formation are evolutionarily linked, suggesting some human amyloids involved in neurodegenerative diseases may share properties with ancient antimicrobial peptides .
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Functional diversity of amyloids: The chameleon properties of Uperin-3.5 demonstrate how structural polymorphism in amyloids can serve regulatory functions, challenging the view of amyloids as exclusively pathological structures .
How can researchers address contradictory results in Uperin-3.5 structural studies?
When faced with seemingly contradictory structural data, researchers should consider:
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Environmental sensitivity: Uperin-3.5 exhibits structural polymorphism depending on conditions such as lipid presence and temperature, so experimental conditions must be carefully controlled and reported .
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Methodological differences: Different structural determination methods (X-ray crystallography, cryo-EM, CD spectroscopy) may capture different conformational states or favor certain structures .
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Dynamic equilibrium: The peptide likely exists in a dynamic equilibrium between conformational states, with experimental conditions shifting this equilibrium .
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Fibril maturation effects: The age of fibrils and formation conditions lead to different structures, so sample history should be documented and considered when interpreting results .
What factors should be considered when designing antimicrobial assays for Uperin-3.5?
When designing antimicrobial assays, researchers should account for:
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Pre-incubation conditions: Whether the peptide is freshly prepared or pre-formed into fibrils significantly affects activity. Pre-formed fibrils (incubated for 5 days) show different activity compared to freshly prepared peptide .
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Environmental factors: The presence of lipids, temperature, and solution conditions all affect secondary structure and antimicrobial activity .
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Time dependence: Rapid aggregation changes effective concentration over time, affecting activity measurements .
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Assay readouts: Both minimum inhibitory concentration (MIC) determination and membrane permeation assays provide complementary information about antimicrobial action .
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Heat treatment effects: Heat treatment of pre-formed fibrils leads to reduced antimicrobial activity (MIC increasing from 4 μM to 16 μM), but may partially reverse upon addition to bacterial cells .
How does the cryo-EM structure of Uperin-3.5 complement crystallographic findings?
Recent cryo-EM studies have revealed the atomic-resolution structure of Uperin-3.5 cross-β fibrils, showing a 3-blade symmetrical propeller arrangement with nine peptides per fibril layer including tight β-sheet interfaces . This structure complements the cross-α fibril conformation determined by crystallography, providing strong evidence for the peptide's conformational versatility and supporting the secondary structure switch mechanism .
What are the implications of Uperin-3.5 research for antimicrobial peptide design?
The findings from Uperin-3.5 research have several implications for the design of novel antimicrobial peptides:
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The importance of environmental responsiveness in regulating antimicrobial activity
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The potential utility of designing peptides with controlled structural transitions
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The possibility of developing peptides that specifically target bacterial membranes
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The potential for creating peptides with reduced susceptibility to heat-induced inactivation
Understanding the molecular details of how Uperin-3.5 switches between different fibril forms could inform the design of synthetic peptides with enhanced stability and activity profiles .
What questions remain unanswered about Uperin-3.5?
Despite significant progress, several questions remain about Uperin-3.5:
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The precise mechanism by which cross-α fibrils disrupt bacterial membranes
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The kinetics of the structural transition between cross-α and cross-β forms
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The complete spectrum of antimicrobial activity against different bacterial species
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The potential for Uperin-3.5 or derivatives as therapeutic antimicrobial agents
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The evolutionary history of Uperin-3.5 and its relationship to other antimicrobial peptides
Addressing these questions will require further structural studies, molecular dynamics simulations, and comprehensive antimicrobial testing against diverse bacterial pathogens .
