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TB-500 Topics

TB-500 Structure & Chemistry

Primary Structure: Amino Acid Sequence

TB-500 consists of 43 amino acids with the following sequence (from N-terminus to C-terminus):

Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu-Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser

Key features of the sequence:

  • N-Terminal Acetylation: The N-terminus is acetylated (Ac-), a post-translational modification important for stability and function
  • Length: 43 amino acids
  • Charged Residues: High content of acidic (Asp, Glu) and basic (Lys) amino acids
  • Proline Residues: Contains 3 proline residues that influence structure
  • Hydrophobic Residues: Contains leucine, isoleucine, and phenylalanine

Molecular Properties

Basic Characteristics

  • Molecular Formula: C₂₁₂H₃₅₀N₅₆O₇₈S
  • Molecular Weight: 4963.44 g/mol
  • Isoelectric Point (pI): Approximately 5.1 (acidic)
  • Net Charge at pH 7: Negative (due to excess acidic residues)
  • Extinction Coefficient: 1490 M⁻¹cm⁻¹ at 280 nm

Amino Acid Composition

TB-500 contains:

  • Glutamic Acid (Glu): 9 residues
  • Lysine (Lys): 7 residues
  • Aspartic Acid (Asp): 4 residues
  • Serine (Ser): 4 residues
  • Threonine (Thr): 3 residues
  • Proline (Pro): 3 residues
  • Glutamine (Gln): 3 residues
  • Isoleucine (Ile): 2 residues
  • Leucine (Leu): 2 residues
  • Alanine (Ala): 2 residues
  • Methionine (Met): 1 residue
  • Phenylalanine (Phe): 1 residue
  • Asparagine (Asn): 1 residue
  • Glycine (Gly): 1 residue

Secondary and Tertiary Structure

Structural Characteristics

TB-500 is classified as an intrinsically disordered protein (IDP), meaning it lacks a stable, well-defined three-dimensional structure in solution. Key structural features include:

  • Flexible Structure: The peptide adopts multiple conformations rather than a single rigid structure
  • α-Helical Tendency: Some regions show propensity to form α-helices, particularly in the presence of actin
  • Extended Conformation: Generally adopts an extended, flexible conformation in solution
  • Conformational Changes: Structure changes upon binding to actin or other partners

Actin-Binding Domain

The central region of TB-500 (approximately residues 5-20) contains the primary actin-binding domain. This region:

  • Adopts a more structured conformation when bound to actin
  • Forms specific contacts with actin's surface
  • Is critical for the peptide's biological activity
  • Shows sequence conservation across species

Chemical Properties

Solubility

  • Water Solubility: Highly soluble in water and aqueous buffers
  • pH Dependence: Solubility varies with pH due to charged residues
  • Salt Effects: Solubility influenced by ionic strength
  • Organic Solvents: Soluble in DMSO, limited solubility in alcohols

Stability

  • pH Stability: Stable across a wide pH range (pH 4-9)
  • Temperature Stability: Relatively stable at room temperature for short periods
  • Oxidation Sensitivity: Methionine residue susceptible to oxidation
  • Proteolytic Stability: Susceptible to degradation by proteases
  • Freeze-Thaw Stability: Can withstand limited freeze-thaw cycles

Chemical Modifications

TB-500 can undergo various chemical modifications:

  • Oxidation: Methionine can be oxidized to methionine sulfoxide
  • Deamidation: Asparagine and glutamine residues can deamidate over time
  • Hydrolysis: Peptide bonds can hydrolyze under harsh conditions
  • Aggregation: Can form aggregates at high concentrations or improper storage

Spectroscopic Properties

UV-Visible Spectroscopy

  • Absorption Maximum: 280 nm (due to phenylalanine)
  • Extinction Coefficient: 1490 M⁻¹cm⁻¹
  • Concentration Determination: Can be quantified by UV absorption

Circular Dichroism (CD)

  • Shows characteristics of disordered structure in solution
  • Increased α-helical content upon actin binding
  • Used to study conformational changes

Binding Properties

Actin Binding

  • Binding Affinity: Kd approximately 0.5-2 μM
  • Stoichiometry: 1:1 binding (one TB-500 per actin monomer)
  • Binding Site: Binds to the barbed end of actin
  • Conformational Change: Both TB-500 and actin undergo conformational changes upon binding

Other Binding Partners

TB-500 may interact with other proteins and molecules:

  • Cell surface receptors (specific receptors not fully characterized)
  • Extracellular matrix components
  • Other cytoskeletal proteins

Structure-Function Relationships

Critical Regions

  • N-Terminal Acetylation: Important for stability and may influence activity
  • Actin-Binding Domain (residues 5-20): Essential for primary biological function
  • C-Terminal Region: May contribute to other biological activities
  • Charged Residues: Influence solubility and protein-protein interactions

Sequence Variants

Natural variants and synthetic modifications have been studied:

  • Truncated versions (e.g., TB4-sulfoxide)
  • Point mutations to study structure-function relationships
  • Modified versions for enhanced stability or activity

Analytical Characterization

Mass Spectrometry

  • Expected Mass: 4963.44 Da
  • Fragmentation Patterns: Can be used to confirm sequence
  • Modification Detection: Can identify oxidation, deamidation, etc.

Chromatographic Properties

  • Reverse-Phase HPLC: Retention time depends on column and gradient
  • Ion Exchange: Binds to cation exchangers at neutral pH
  • Size Exclusion: Elutes according to molecular weight

Comparison with Related Peptides

Compared to BPC-157 (15 amino acids, 1419.56 g/mol), TB-500 is significantly larger and more complex. GHK-Cu (3 amino acids, 404.0 g/mol) is much smaller and simpler. TB-500's larger size contributes to its more complex biological activities but also makes it more challenging to synthesize and characterize.

Conclusion

TB-500 is a 43-amino acid peptide with a molecular weight of 4963.44 g/mol. Its structure is characterized by intrinsic disorder, with flexibility that allows it to adopt different conformations depending on its environment and binding partners. The peptide's primary biological activity stems from its ability to bind actin through a central binding domain. Understanding TB-500's chemical and structural properties is essential for proper handling, storage, and appreciation of its biological mechanisms.

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