Recombinant Uperoleia inundata Uperin-2.1

What is Uperin-2.1 and how was it first isolated from Uperoleia inundata?

Uperin-2.1 is a bioactive peptide first isolated from the dorsal glandular secretions of the Australian floodplain toadlet Uperoleia inundata . The isolation process typically involves mild electrical stimulation of the dorsal glands to collect skin secretions, followed by fractionation using reversed-phase HPLC. Initial identification is conducted through mass spectrometry techniques such as MALDI-TOF MS, which establishes the molecular weight, followed by Edman degradation sequencing to determine the amino acid sequence. For peptides with post-translational modifications at the N-terminus (as is common with some amphibian peptides), electrospray mass spectrometry using both negative and positive ion modes is employed to identify modifications such as pyroglutamic acid or sulfonation .

What are the structural characteristics of Uperin-2.1?

Uperin-2.1 belongs to the family of amphibian antimicrobial peptides that can adopt different secondary structure conformations depending on environmental conditions. Similar to other uperin peptides such as uperin 3.5, Uperin-2.1 likely undergoes conformational transitions between α-helical and β-sheet structures . The peptide contains an amphipathic region that facilitates interaction with biological membranes. When examining related uperin peptides through cryo-EM, they form amyloid cross-β fibrils with mated β-sheets at atomic resolution . The amidated C-terminus, common in amphibian antimicrobial peptides, enhances stability through inter-strand hydrogen bonds .

How does the expression of Uperin-2.1 vary seasonally in Uperoleia inundata?

Based on observations of related amphibian antimicrobial peptides, the expression of skin peptides like Uperin-2.1 likely exhibits seasonal variation. Similar amphibian species such as Litoria splendida and Litoria rothii demonstrate significant seasonal changes in the composition of their skin secretions . During breeding seasons, the expression of bioactive peptides typically increases, while declining during non-breeding periods. The mechanisms regulating this seasonal variation involve hormonal control, particularly by reproductive hormones. Environmental factors such as temperature, humidity, and photoperiod also influence peptide expression patterns . This seasonal variation represents an important adaptation to changing environmental conditions and potential pathogen exposure.

What are the optimal conditions for recombinant expression of Uperin-2.1, and how do different expression systems affect its structural integrity?

For recombinant expression of amphibian antimicrobial peptides like Uperin-2.1, several expression systems may be employed with varying advantages:

• E. coli expression system: This is the most commonly used approach, typically utilizing pET vectors with fusion tags (such as His6, SUMO, or thioredoxin) to mitigate the toxicity of antimicrobial peptides to the host cells. Optimal conditions include:

  • Induction with 0.5-1.0 mM IPTG

  • Expression temperature of 20-25°C rather than 37°C to enhance proper folding

  • Addition of 1% glucose to the medium to prevent leaky expression

  • Use of BL21(DE3)pLysS strain to control background expression

• Yeast expression systems: Pichia pastoris and Saccharomyces cerevisiae can be advantageous for peptides requiring post-translational modifications.

• Cell-free expression: This avoids toxicity issues completely and allows direct synthesis of difficult peptides.

The expression system significantly impacts structural integrity. E. coli systems may result in inclusion body formation requiring refolding steps, potentially affecting the native structure. Yeast systems typically produce correctly folded peptides but may introduce unwanted glycosylation. The most reliable approach for confirming structural integrity post-expression is comparative analysis using circular dichroism spectroscopy, comparing the recombinant peptide with the native form isolated from amphibian secretions.

How do the cross-β fibril conformations of Uperin-2.1 compare to those of other uperin peptides, and what implications do these structural differences have for antimicrobial function?

The cross-β fibril conformations of uperin peptides demonstrate unique structural arrangements that directly influence their antimicrobial function. Comparative analysis of uperin peptide structures reveals:

• Structural organization: While specific data on Uperin-2.1 fibrils is limited in the provided search results, related uperin peptides such as uperin 3.5 form complex cross-β structures. Uperin 3.5 forms a distinctive 3-blade symmetrical propeller arrangement with nine peptides per fibril layer, featuring tight β-sheet interfaces . This arrangement creates a positively charged and hydrophobic core composed of inward-facing Arg7 and Leu5 residues.

• Conformational flexibility: Uperin peptides can adopt both cross-α and cross-β conformations, suggesting a secondary structure switch mechanism that may be crucial for membrane interaction and disruption .

• Functional implications: The specific cross-β arrangements influence antimicrobial activity through:

  • Membrane interaction capabilities - the distribution of charged residues on the fibril surface determines how the peptide interacts with bacterial membranes

  • Oligomerization potential - the propensity to form specific fibril structures affects the peptide's ability to create membrane pores

  • Stability in biological environments - the tight packing in cross-β structures provides resistance against proteolytic degradation

These structural characteristics likely determine target specificity, potency, and the mechanism of antimicrobial action (membrane disruption vs. intracellular targets).

What mechanisms underlie the potential synergistic effects between Uperin-2.1 and other antimicrobial peptides in amphibian skin secretions?

• Complementary membrane targeting: Different peptides may target distinct components of microbial membranes. While some peptides target negatively charged phospholipids, others may interact preferentially with neutral lipids or specific membrane proteins. Amphibian skin secretions typically contain multiple peptides with varying physiochemical properties, creating a multi-modal attack on pathogen membranes .

• Sequential action mechanisms: Research on amphibian peptides suggests a coordinated temporal action where:

  • Initial membrane destabilization by one peptide class facilitates the entry or action of secondary peptides

  • Peptides with rapid kinetics create membrane disturbances that allow slower-acting peptides to reach intracellular targets

• Inhibition of resistance mechanisms: Multiple peptides with different structural properties prevent the development of microbial resistance. When bacteria develop resistance to one peptide structure (e.g., α-helical peptides), they remain susceptible to peptides with β-sheet conformations.

• Concentration-dependent conformational switching: Uperin peptides demonstrate the ability to switch between different secondary structures (α-helical and β-sheet) depending on concentration and environmental conditions . This conformational plasticity enables dynamic responses to different microbial challenges.

This synergistic action explains why amphibian skin secretions contain a complex mixture of bioactive peptides rather than a single dominant antimicrobial compound .

What are the optimal protocols for solid-phase synthesis of Uperin-2.1 and what challenges must be addressed during chemical synthesis?

The optimal protocol for solid-phase synthesis of Uperin-2.1 involves Fmoc chemistry with the following methodological considerations:

• Resin selection: Rink amide MBHA resin is preferred for C-terminally amidated peptides common in amphibian antimicrobial peptides . This ensures the natural amidated C-terminus found in native Uperin-2.1.

• Coupling conditions:

  • HBTU/DIPEA activation with 4 equivalents of Fmoc-protected amino acids

  • Extended coupling times (90-120 minutes) for difficult residues (β-branched amino acids like Ile, Val, Thr)

  • Double coupling for problematic sequences, particularly in hydrophobic regions

• Deprotection strategy:

  • 20% piperidine in DMF for Fmoc removal

  • Monitoring deprotection efficiency using conductivity measurements

• Cleavage conditions:

  • TFA/TIS/H2O (95:2.5:2.5) mixture for 3 hours at room temperature

  • For peptides containing Trp or Met residues, addition of 2.5% EDT to prevent oxidation

• Challenges and solutions:

• Purification strategy:

  • Reversed-phase HPLC using C18 columns with shallow gradients (1% ACN/minute)

  • MALDI-TOF MS confirmation of molecular weight

  • Analytical HPLC to confirm >95% purity before biological testing

How can researchers effectively analyze the binding kinetics between Uperin-2.1 and bacterial membrane components using surface plasmon resonance?

Surface plasmon resonance (SPR) analysis of Uperin-2.1 binding to bacterial membrane components requires a systematic approach:

• Membrane model preparation:

  • Liposome capture approach: L1 sensor chips coated with phospholipid liposomes mimicking bacterial membranes (typically POPE/POPG at 7:3 ratio for Gram-negative bacteria or POPG/cardiolipin at 8:2 ratio for Gram-positive bacteria)

  • Hybrid bilayer approach: HPA chips with alkylated surface coupled with a phospholipid monolayer

• Experimental design:

  • Multi-cycle kinetics: Series of increasing peptide concentrations (typically 0.1-50 μM) injected over immobilized membrane

  • Single-cycle kinetics: Continuous increasing concentration without regeneration steps

  • Temperature control at 25°C to maintain membrane fluidity

• Data analysis methodology:

  • Initial binding rates analysis to determine association rate constants (ka)

  • Steady-state analysis for dissociation constants (KD)

  • Two-state binding model to account for initial surface binding followed by membrane insertion

• Control experiments:

  • Parallel analysis using mammalian-like membranes (POPC/cholesterol) to determine selectivity index

  • Non-antimicrobial peptide controls with similar physicochemical properties

  • Testing at multiple ionic strengths to distinguish electrostatic from hydrophobic interactions

• Addressing technical challenges:

  • Non-specific binding to sensor surfaces minimized by addition of 0.005% P20 surfactant

  • Mass transport limitations addressed by lower ligand density and higher flow rates (50 μL/min)

  • Reference subtraction using blank liposome surfaces or scrambled peptide controls

• Data interpretation framework:

  • Concentration-dependent changes in binding profiles indicate potential conformational changes or oligomerization

  • Biphasic dissociation curves suggest membrane insertion following initial binding

  • Comparison with cross-β fibril formation propensity to correlate structure with membrane binding kinetics

What analytical techniques are most effective for characterizing the folding dynamics of Uperin-2.1 during membrane interaction?

Multiple complementary analytical techniques are required to fully characterize the folding dynamics of Uperin-2.1 during membrane interaction:

• Time-resolved circular dichroism (CD) spectroscopy:

  • Provides real-time monitoring of secondary structure transitions

  • Rapid-mixing stopped-flow CD can capture millisecond transitions between random coil, α-helical, and β-sheet conformations

  • Quantitative analysis of spectral changes at 208 nm, 222 nm, and 218 nm wavelengths correlate with α-helical and β-sheet content respectively

• Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR):

  • Monitors amide I band (1600-1700 cm^-1) changes during membrane binding

  • Can distinguish between different β-sheet arrangements (parallel vs. antiparallel) based on peak positions

  • Particularly valuable for detecting cross-β fibril formation characteristic of uperin peptides

• Thioflavin T (ThT) fluorescence kinetics:

  • Real-time monitoring of amyloid-like cross-β structure formation

  • Characteristic sigmoidal curve indicates nucleation-dependent fibril formation

  • Correlation with antimicrobial activity onset provides mechanistic insights

• Advanced fluorescence techniques:

  • Intrinsic tryptophan fluorescence for tertiary structure changes

  • Site-specific FRET pairs to measure intramolecular distances during folding

  • Time-resolved anisotropy to quantify rotational diffusion changes during membrane insertion

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps regions of secondary structure formation during membrane binding

  • Identifies protected backbone amide hydrogens indicating structured regions

  • Provides residue-specific folding information not available from spectroscopic methods

• Solid-state NMR with model membranes:

  • Determines precise orientation and depth of insertion in lipid bilayers

  • Identifies specific residues involved in membrane interaction

  • Provides atomic-level details of conformational changes

• Cryo-electron microscopy:

  • Directly visualizes peptide assembly states on membrane surfaces

  • Can capture intermediate oligomeric states during fibril formation

  • Allows correlation between cross-β structure formation and membrane disruption

This multi-technique approach provides a comprehensive understanding of the conformational transitions between unstructured, α-helical, and cross-β states during the antimicrobial activity of Uperin-2.1.

How does the amino acid sequence of Uperin-2.1 influence its propensity to form cross-β fibril structures, and what residues are critical for this function?

The amino acid sequence of Uperin-2.1 contains specific features that govern its propensity to form cross-β fibril structures:

• Key sequence determinants:

  • Alternating hydrophobic and hydrophilic residues create an amphipathic pattern conducive to β-sheet formation

  • Presence of aromatic residues (particularly phenylalanine) that promote π-stacking interactions along the fibril axis

  • Strategic positioning of basic residues (lysine and arginine) that can form salt bridges stabilizing inter-sheet interactions

• Critical residues for fibril formation: Based on structural studies of related uperin peptides, several residue types play crucial roles:

  • Arginine residues: In uperin 3.5, Arg7 forms part of a positively charged and hydrophobic core that stabilizes the propeller-like fibril arrangement

  • Leucine residues: Hydrophobic residues like Leu5 in uperin 3.5 create tightly mated β-sheet interfaces through van der Waals interactions

  • Asparagine residues: Form inter-sheet hydrogen bonds that stabilize the mated β-sheet structure, as seen with Asn15 in uperin 3.5

  • C-terminal amidation: The amidated C-terminus forms important inter-strand hydrogen bonds along the fibril axis, connecting adjacent peptide chains

• Structure-function relationships:

  • The arrangement of positively charged residues on the fibril surface determines interaction with negatively charged bacterial membranes

  • Hydrophobic residues positioned in the core of the fibril provide stability while those on the surface mediate membrane penetration

  • Small, flexible residues like glycine enable the sharp turns necessary for compact fibril packing

• Sequence regions with distinct roles:

  • N-terminal region: Often more flexible, may remain partially unstructured even in the fibril state

  • Central core: Forms the primary β-strand regions involved in the cross-β spine

  • C-terminal region: Often contains basic residues that enhance antimicrobial activity through electrostatic interactions with bacterial membranes

Understanding these sequence-structure relationships is essential for designing synthetic variants with enhanced stability or targeted antimicrobial activity.

What experimental approaches can distinguish between the membrane disruption mechanisms of Uperin-2.1 in different bacterial species?

Distinguishing between membrane disruption mechanisms requires a multi-parametric experimental approach:

• Dye leakage assays with liposome models:

  • Large unilamellar vesicles (LUVs) with composition mimicking different bacterial membranes

  • Encapsulation of size-graded fluorescent markers (calcein, FITC-dextrans of varying molecular weights)

  • Kinetic analysis of leakage patterns:

    • All-or-none mechanism: Complete release from affected vesicles

    • Graded mechanism: Partial release from all vesicles

    • Size-dependent leakage: Indicates specific pore dimensions

• Electron microscopy visualization:

  • Negative staining TEM of bacteria after peptide treatment at sub-lethal concentrations

  • Cryo-EM of model membranes with peptide to visualize membrane deformations

  • Quantitative analysis of:

    • Membrane thinning

    • Bleb formation

    • Pore structures

    • Carpet-like aggregation patterns

• Electrophysiology approaches:

  • Planar lipid bilayer conductance measurements

  • Patch-clamp analysis of peptide-induced channels

  • Determination of:

    • Single-channel conductance

    • Ion selectivity

    • Voltage dependence

    • Channel lifetime and flickering behavior

• Atomic force microscopy (AFM):

  • Real-time visualization of membrane topography changes

  • Force spectroscopy to measure membrane elasticity alterations

  • High-speed AFM to capture kinetics of membrane disruption

• Differential mechanistic analysis across bacterial species:

• Genetic approaches:

  • Bacterial strains with altered membrane composition

  • Resistance mechanisms analysis through transcriptomics

  • Correlation between membrane composition and susceptibility pattern

This comprehensive approach enables detailed characterization of how Uperin-2.1 disrupts membranes across different bacterial species and provides insight into the structure-function relationship of its antimicrobial mechanisms.

How can molecular dynamics simulations be effectively employed to predict the interaction between Uperin-2.1 fibrils and bacterial membranes?

Molecular dynamics (MD) simulations provide powerful insights into Uperin-2.1 fibril-membrane interactions through a systematic computational approach:

• Simulation system construction:

  • Fibril model building based on cryo-EM structural data of related uperin peptides

  • Bacterial membrane model construction with appropriate lipid composition:

    • E. coli-like membranes: POPE/POPG (7:3) bilayers

    • S. aureus-like membranes: POPG/cardiolipin (8:2) bilayers

  • System solvation with explicit water molecules and physiological ion concentration (0.15 M NaCl)

• Simulation protocol optimization:

  • Multi-stage equilibration procedure:

    • Position-restrained equilibration of lipids around rigid fibril (10-20 ns)

    • Gradual release of positional restraints (50-100 ns)

    • Production simulations (1-5 μs) using specialized hardware (GPU acceleration)

  • CHARMM36m or AMBER ff14SB force fields for accurate peptide-lipid interactions

  • Enhanced sampling techniques (umbrella sampling, metadynamics) to overcome energy barriers

• Analytical framework:

  • Quantitative metrics for membrane disruption:

    • Local membrane thinning measurements

    • Lipid order parameter calculations

    • Water penetration depth profiles

    • Electrostatic potential maps across the membrane

  • Peptide-lipid interaction analysis:

    • Hydrogen bond persistence between specific residues and lipid headgroups

    • Salt bridge formation between basic residues and anionic lipids

    • Hydrophobic contacts between nonpolar residues and lipid tails

  • Fibril stability and conformational dynamics:

    • RMSD/RMSF calculations for fibril backbone stability

    • Secondary structure persistence analysis

    • Inter-strand hydrogen bond occupancy

• Advanced simulation approaches:

  • Coarse-grained simulations (MARTINI force field) for longer timescales (10-100 μs)

  • Hybrid quantum mechanics/molecular mechanics (QM/MM) for specific interactions

  • Constant-pH simulations to model pH-dependent conformational changes

• Validation and prediction framework:

  • Correlation of simulation predictions with experimental observables:

    • Fluorescence leakage kinetics

    • Lipid extraction rates

    • Membrane elastic properties

  • Design of point mutations based on simulation insights:

    • Identify critical residues for membrane binding

    • Predict effects of sequence modifications on fibril-membrane interactions

    • Guide rational design of enhanced antimicrobial variants

This computational approach provides atomic-level insights into the mechanisms by which Uperin-2.1 fibrils interact with and disrupt bacterial membranes, complementing experimental studies and guiding future peptide design efforts.