A. Draw Ramachandran plot and explain it importance. B. Explain the N-terminal protein sequencing method

Ramachandran Plot and N-terminal Protein Sequencing Method

Part A: Ramachandran Plot

Definition:

A Ramachandran plot is a graphical representation that shows the possible combinations of dihedral angles (phi φ and psi ψ) of amino acids in a protein structure. It helps visualize allowed and disallowed conformations due to steric hindrance.

Diagram:

Importance:

• Helps in validating protein 3D structures obtained from X-ray crystallography or NMR.
• Identifies regions with favorable backbone conformations.
• Used to assess the quality of protein models in structural biology.

Allowed Regions:

• Alpha helix: φ ≈ -60°, ψ ≈ -40°
• Beta sheet: φ ≈ -120°, ψ ≈ 120°
• Left-handed helix: φ ≈ 60°, ψ ≈ 60°

Part B: N-terminal Protein Sequencing Method

Definition:

The N-terminal sequencing method identifies the sequence of amino acids starting from the amino (N) end of a protein. The most common method used is Edman degradation.

Steps in Edman Degradation:

1. Labeling: The N-terminal amino acid reacts with phenyl isothiocyanate (PITC) under alkaline conditions to form a phenylthiocarbamoyl (PTC) derivative.
2. Cleavage: The PTC-amino acid is selectively cleaved without breaking other peptide bonds.
3. Identification: The cleaved amino acid is converted into a stable phenylthiohydantoin (PTH) derivative and identified using chromatography or electrophoresis.
4. Repeat: The cycle repeats for 20–30 residues to determine the N-terminal sequence.

Advantages:

• Provides direct information about the amino acid sequence
• Useful for identifying unknown proteins

Limitations:

• Cannot sequence beyond 30–50 residues efficiently
• Blocked N-termini (e.g., acetylated) must be modified before sequencing

Conclusion

Both the Ramachandran plot and N-terminal sequencing are fundamental tools in protein biochemistry. The plot is essential for understanding protein folding and structural validation, while N-terminal sequencing helps determine the identity and structure of proteins. Together, they enhance our understanding of protein structure and function.