21. The exact backbone dihedral angles in a folded protein can be obtained by

    (1) deconvolution of its circular dichroism spectra obtained at different pH and temperature
    (2) estimating the number of protons that exchange with deuterium on treating the protein with D₂O
    (3) forming fibres of the protein and analyzing the fibre diffraction pattern
    (4) analysis of the crystal structure of the protein obtained by X-ray diffraction at high resolutions

Determining Backbone Dihedral Angles in Folded Proteins

The exact backbone dihedral angles (ϕ and Ψ) in a folded protein provide crucial insights into its secondary structure and stability. These angles define the conformation of the protein backbone and are determined using advanced structural biology techniques.

Correct Answer:

The correct option is:

(4) Analysis of the crystal structure of the protein obtained by X-ray diffraction at high resolutions.

Explanation of Techniques Used to Determine Dihedral Angles

1. X-ray Crystallography (Correct Answer)

• Most accurate method for obtaining atomic-level details of proteins.
• Provides high-resolution structural data, allowing precise measurement of ϕ and Ψ angles.
• Requires crystallization of the protein, followed by diffraction analysis.

2. Circular Dichroism (CD) Spectroscopy

• Used to estimate secondary structure composition.
• Involves deconvolution of spectra at different pH and temperatures.
• Cannot provide exact backbone dihedral angles.

3. Hydrogen-Deuterium Exchange (HDX) with D₂O

• Determines protein dynamics and stability.
• Measures proton exchange rates, not backbone dihedral angles.

4. Fiber Diffraction Analysis

• Used for fibrillar proteins like collagen and amyloids.
• Provides general structural insights, but not precise dihedral angles.

Biological Significance of Backbone Dihedral Angles

1. Protein Folding and Stability

• ϕ and Ψ angles define secondary structures like α-helices and β-sheets.
• Deviations from allowed angles can lead to misfolding and aggregation.

2. Ramachandran Plot Validation

• Helps in validating protein structures by analyzing conformationally allowed regions.
• Essential for protein modeling and structural prediction.

3. Drug Discovery and Molecular Modeling

• Knowing precise backbone angles helps in designing small molecule inhibitors.
• Structural data aids in computational simulations for drug-target interactions.

Conclusion

The most accurate method to determine backbone dihedral angles in folded proteins is X-ray crystallography, as it provides high-resolution structural data. Other techniques like CD spectroscopy, HDX, and fiber diffraction offer complementary insights but do not directly measure dihedral angles.