SNCA 96-140 Human

What is SNCA 96-140 Human and how does it relate to full-length alpha-synuclein?

SNCA 96-140 Human is a recombinant deletion mutant containing only amino acids 96-140 of the full-length alpha-synuclein protein. The full alpha-synuclein protein (αSyn-140) consists of 140 amino acids and is encoded by the SNCA gene. The 96-140 fragment represents the C-terminal acidic tail of the protein, which has been shown to possess important regulatory functions. This segment is particularly significant as recent studies have demonstrated that alpha-synuclein's chaperone activity is lost upon removing this C-terminal acidic tail (amino acids 96-140) .

The amino acid sequence of SNCA 96-140 is: MKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA, with an additional methionine attached at the N-terminus when produced recombinantly . The fragment has a theoretical molecular mass of approximately 5,217 Da, significantly smaller than the full-length protein .

Why is the C-terminal region (96-140) particularly important in alpha-synuclein research?

The C-terminal region of alpha-synuclein plays several critical roles in the protein's function and pathological behavior:

• Regulatory function: The C-terminal region appears to regulate the aggregation propensity of the full-length protein. Studies suggest that this acidic tail influences conformational dynamics of alpha-synuclein.

• Chaperone activity: Recent research has shown that alpha-synuclein possesses chaperone activity that is specifically lost when the C-terminal acidic tail (amino acids 96-140) is removed . This indicates a functional importance beyond mere structural roles.

• Metal binding: The C-terminal region contains numerous negatively charged residues that participate in metal binding, particularly with divalent cations like cobalt and manganese, which may influence aggregation behavior .

• Pathological implications: Understanding the specific roles of the 96-140 region helps elucidate how different alpha-synuclein domains contribute to pathological aggregation in Parkinson's disease and related synucleinopathies .

How do alpha-synuclein isoforms differ structurally and functionally?

Alpha-synuclein exists in several isoforms generated through alternative splicing of the SNCA gene, which comprises six exons. The primary isoforms include:

Research has demonstrated that αSyn-112 and αSyn-98 exhibit significantly faster aggregation kinetics compared to the full-length αSyn-140. Even small amounts of these faster-aggregating isoforms can accelerate the aggregation of αSyn-140, reducing aggregation half-time . This finding has important implications for understanding how alternative splicing might contribute to pathological processes in synucleinopathies.

What are the optimal storage and handling conditions for SNCA 96-140?

For reliable experimental outcomes when working with SNCA 96-140, follow these storage and handling guidelines:

Storage conditions:

• Store at -20°C for long-term preservation

• For short-term use (2-4 weeks), store at 4°C

• Add a carrier protein (0.1% HSA or BSA) for long-term storage to enhance stability

• Avoid multiple freeze-thaw cycles which can affect protein integrity

Handling recommendations:

• Work with the protein in 20mM Tris-HCl buffer (pH 7.4-7.5) containing 100mM NaCl to maintain a monomeric starting material

• Use sterile technique when handling the lyophilized powder

• Reconstitute immediately before use rather than storing reconstituted protein for extended periods

• Document batch information, as batch-to-batch consistency is essential for reproducible results

How should researchers design aggregation studies using SNCA 96-140?

When designing aggregation studies with SNCA 96-140, consider these methodological approaches:

• Buffer selection: Use 20mM Tris-HCl buffer with 100mM NaCl at pH 7.4-7.5 as the starting condition, as this composition ensures a highly monomeric starting material .

• Protein concentration: Carefully control protein concentration, as aggregation kinetics are concentration-dependent. Typical working concentrations range from 35-70 μM for reproducible kinetics.

• Agitation conditions: Standardize agitation parameters (e.g., constant stirring at 37°C or shaking at 200-500 rpm) as these significantly affect aggregation rates.

• Monitoring techniques: Employ multiple complementary techniques to track aggregation:

  • Thioflavin T fluorescence for β-sheet formation kinetics

  • Dynamic light scattering for particle size distribution

  • Transmission electron microscopy for morphological characterization

  • Circular dichroism for secondary structure changes

• Time points: Establish appropriate time points based on preliminary experiments, as SNCA 96-140 may aggregate with different kinetics than full-length protein.

• Controls: Include controls such as non-aggregating protein variants or known aggregation inhibitors to validate experimental setup.

What analytical techniques provide the most informative data for SNCA 96-140 structural studies?

Several complementary analytical techniques are essential for comprehensive structural characterization of SNCA 96-140:

• Native top-down mass spectrometry and ion mobility MS: These techniques have proven valuable for characterizing metal binding properties of alpha-synuclein, particularly interactions with cobalt and manganese ions . They provide insights into conformational changes induced by metal binding.

• Transmission electron microscopy (TEM): Essential for visualizing aggregate morphologies and distinguishing between different types of assemblies (e.g., oligomers, protofibrils, mature fibrils).

• Homology modeling: Computer-based structural prediction using experimentally determined structures of related proteins. This approach is particularly useful given the intrinsically disordered nature of alpha-synuclein in its native state .

• Circular dichroism (CD) spectroscopy: Monitors secondary structure changes during aggregation, particularly the transition from random coil to β-sheet structure.

• Fourier-transform infrared spectroscopy (FTIR): Provides detailed information about secondary structure components and can detect subtle differences in β-sheet arrangements.

• Nuclear magnetic resonance (NMR) spectroscopy: Offers atomic-level structural information, especially useful for characterizing the monomeric form and early oligomeric species.

How can co-aggregation studies with SNCA 96-140 and full-length alpha-synuclein be optimized?

Co-aggregation studies investigating interactions between SNCA 96-140 and full-length alpha-synuclein require careful experimental design:

• Ratio determination: Test various molar ratios of SNCA 96-140 to full-length protein. Research indicates that even small amounts of certain isoforms can significantly accelerate aggregation of αSyn-140, so start with ratios ranging from 1:100 to 1:10 .

• Labeling strategies: Consider differential labeling approaches (e.g., fluorescent tags or isotopic labeling) to distinguish the behavior of each component within the mixture. Ensure labels don't significantly alter aggregation properties.

• Kinetic analysis: Implement mathematical models to quantify aggregation parameters:

  • Lag time

  • Growth rate

  • Half-time (t50)

  • Maximum aggregation

• Cross-seeding protocols:

  • Pre-form seeds from SNCA 96-140 under controlled conditions

  • Sonicate seeds to standardize size

  • Add seeds at defined concentrations to monomeric full-length protein

  • Monitor aggregation with techniques like Thioflavin T fluorescence

• Control conditions: Include parallel experiments with:

  • SNCA 96-140 alone

  • Full-length alpha-synuclein alone

  • Non-interacting protein controls

• Data analysis: Apply appropriate kinetic models to determine whether interactions are synergistic, additive, or inhibitory.

What is known about how post-translational modifications affect SNCA 96-140 behavior?

The C-terminal region of alpha-synuclein (96-140) contains multiple sites for post-translational modifications (PTMs) that significantly alter its properties:

• Phosphorylation: Several serine and tyrosine residues in the 96-140 region can be phosphorylated, including:

  • Ser129 (most studied modification in alpha-synuclein)

  • Tyr125, Tyr133, and Tyr136

  Phosphorylation generally inhibits fibril formation and alters interactions with cellular components.

• Truncation: C-terminally truncated forms of alpha-synuclein show enhanced aggregation propensity compared to the full-length protein. The truncation removes the inhibitory effect of the C-terminal region on aggregation.

• Oxidation: Methionine residues in the C-terminal region are susceptible to oxidation, which can alter protein conformation and aggregation properties.

• Metal binding: The acidic C-terminal region binds divalent metal ions such as copper, iron, cobalt, and manganese, which can influence aggregation behavior. Native top-down mass spectrometry and ion mobility MS have been used to characterize these metal-binding properties .

• Ubiquitination: Lysine residues in this region can be ubiquitinated, affecting protein clearance and degradation pathways.

Experimental approaches to study these modifications include:

• Site-directed mutagenesis to mimic or prevent specific PTMs

• In vitro enzymatic modification systems

• Mass spectrometry to identify and quantify PTMs

• Comparative aggregation studies between modified and unmodified proteins

How does SNCA 96-140 interact with cellular membranes compared to full-length alpha-synuclein?

The interaction of SNCA 96-140 with cellular membranes differs significantly from that of full-length alpha-synuclein:

What are common pitfalls in experimental design when working with SNCA 96-140?

Researchers should be aware of several common challenges when designing experiments with SNCA 96-140:

• Aggregation variability: SNCA 96-140 aggregation kinetics can be highly sensitive to experimental conditions. Control these variables:

  • Precise protein concentration (verify by multiple methods)

  • Consistent buffer composition and pH

  • Temperature fluctuations (maintain at exactly 37°C)

  • Identical agitation conditions between experiments

• Batch-to-batch variation: Different preparations may show varied behavior despite identical protocols. Mitigation strategies include:

  • Using single batches for comparative experiments

  • Characterizing each batch thoroughly before use

  • Including internal standards for normalization

• Protein adsorption to surfaces: The protein may adsorb to plastic surfaces, changing effective concentration. Consider:

  • Using low-binding tubes and plates

  • Adding small amounts of carrier protein when appropriate

  • Consistent equilibration times in new containers

• Contaminant effects: Trace contaminants can dramatically affect aggregation. Ensure:

  • High purity standards (>95% by SDS-PAGE)

  • Filtration of all buffers

  • Use of high-quality water and reagents

• Inappropriate controls: Failing to include proper controls can lead to misinterpretation. Always include:

  • Non-aggregating protein controls

  • Buffer-only controls

  • Appropriate positive controls

How can researchers enhance reproducibility in SNCA 96-140 aggregation studies?

To maximize reproducibility in SNCA 96-140 aggregation studies, implement these methodological approaches:

• Standardized protein preparation:

  • Use recombinant protein with verified purity (>95% by SDS-PAGE)

  • Implement consistent expression and purification protocols

  • Verify protein identity by mass spectrometry

  • Aliquot proteins to minimize freeze-thaw cycles

• Detailed protocol documentation:

  • Record precise buffer compositions including exact pH values

  • Document all material sources and lot numbers

  • Specify equipment models and settings

  • Time each step consistently

• Environmental control:

  • Use temperature-controlled incubators with minimal fluctuation

  • Shield experiments from light when appropriate

  • Maintain consistent humidity levels

  • Position samples identically within equipment

• Data collection standardization:

  • Establish fixed time points for measurements

  • Use the same instrument settings across experiments

  • Implement automated data collection when possible

  • Include internal standards for instrument performance

• Statistical robustness:

  • Perform sufficient technical and biological replicates (minimum n=3)

  • Apply appropriate statistical tests

  • Report variability metrics (standard deviation, confidence intervals)

  • Consider power analysis for sample size determination

What are the key considerations when comparing results across different alpha-synuclein fragments?

When comparing experimental results between SNCA 96-140 and other alpha-synuclein fragments or isoforms, researchers should consider these important factors:

How is SNCA 96-140 being utilized in therapeutic development for synucleinopathies?

SNCA 96-140 plays a critical role in several therapeutic development approaches for Parkinson's disease and related synucleinopathies:

• Antibody development:

  • Antibodies targeting the C-terminal region can aid in clearance of extracellular alpha-synuclein

  • These approaches aim to prevent cell-to-cell transmission of aggregates

  • C-terminal-specific antibodies may have different effects than those targeting other regions

• Small molecule screening:

  • SNCA 96-140 serves as a model system for screening compounds that might inhibit aggregation

  • Compounds like epigallocatechin gallate (EGCG) have been shown to remodel alpha-synuclein fibrils

  • The C-terminal fragment allows researchers to target region-specific interactions

• Peptide inhibitors:

  • Peptides designed to interact with the C-terminal region may disrupt harmful protein-protein interactions

  • These can serve as lead compounds for drug development

  • Testing against SNCA 96-140 provides mechanistic insights about binding specificity

• Chaperone mimetics:

  • Since the C-terminal region (96-140) is associated with chaperone activity , molecules that enhance or mimic this function are being explored

  • These approaches aim to prevent misfolding rather than targeting aggregates after formation

What role does the 96-140 region play in alpha-synuclein's interaction with other proteins?

The C-terminal region of alpha-synuclein (96-140) mediates numerous protein-protein interactions crucial for both normal function and pathological processes:

• Chaperone activity interactions:

  • The 96-140 region is essential for alpha-synuclein's chaperone activity

  • This activity influences interactions with misfolded proteins

  • Loss of this region eliminates the protein's ability to function as a chaperone

• Protein degradation pathway interactions:

  • The C-terminal region contains recognition sites for various cellular degradation machinery

  • These interactions influence alpha-synuclein clearance and turnover

  • Altered degradation may contribute to pathological accumulation

• Synaptic protein interactions:

  • As a presynaptic protein, alpha-synuclein's C-terminal region mediates contacts with vesicular transport components

  • These interactions influence neurotransmitter release and synaptic function

  • Disruption may contribute to early synaptic dysfunction in disease states

• Experimental approaches to study these interactions:

  • Yeast two-hybrid screening to identify interaction partners

  • Co-immunoprecipitation assays with SNCA 96-140 vs. full-length protein

  • Proximity labeling methods (BioID, APEX) to identify in vivo interaction networks

  • Cross-linking mass spectrometry to map specific binding interfaces

How does evolutionary conservation of the 96-140 region inform functional studies?

Evolutionary analysis of the SNCA gene provides valuable insights into the functional significance of the 96-140 region:

• Conservation patterns:

  • The SNCA gene shows varied conservation across sarcopterygians (lobe-finned fish and tetrapods)

  • Different regions of alpha-synuclein show distinct evolutionary pressures

  • Analysis of these patterns can highlight functionally critical domains

• Species-specific variations:

  • Comparative analysis of the 96-140 region across species reveals:

    • Highly conserved motifs likely essential for basic function

    • Variable regions that may relate to species-specific adaptations

    • Correlation between conservation and disease-associated mutations

• Functional implications:

  • Highly conserved residues in the 96-140 region likely serve crucial physiological roles

  • These residues are prime targets for functional studies and therapeutic development

  • Evolutionary analysis can help distinguish between pathological changes and normal variations

• Research applications:

  • Design of cross-species comparative studies to test functional hypotheses

  • Identification of suitable model organisms for specific aspects of alpha-synuclein biology

  • Improved interpretation of genetic variants identified in patient populations

By understanding the evolutionary context of the 96-140 region, researchers can better focus functional studies on the most biologically significant aspects of this protein domain.