BMOL2201 Tutorial 2

2. Definition and Importance of Proteins

• Proteins are polymers of amino acids, performing essentialcellular functions (enzyme catalysis, transport, structural support, signaling).

• Key questions:

  • What differentiates proteins?

  • How do sequence variations affect function?

3. Primary Structure of Proteins

• Order of amino acids (N to C direction).

• Amino acids are referred to as residues.

4. Protein Sequencing Workflow

1. Remove disulfide bonds.

2. Fragment peptide using multiple methods.

3. Sequence using Edman degradation or mass spectrometry.

4. Reconstruct protein from overlapping sequences.

5. Analyze with disulfide bonds intact.

5. Identification of N- and C-Termini

• Amino terminus: Sanger method or Dansyl Chloride.

• Carboxyl terminus: Carboxypeptidase, followed by HPLC for residue determination.

6. Fragmentation of Peptides with Proteases

• Proteases cut after specific residues:

  • Trypsin: Cuts after K & R.

  • Chymotrypsin: Cuts after F, Y & W.

  • Cyanogen bromide: Cuts after M.

7. Edman Degradation for Sequencing

• Identifies one residue at a time from N-terminus; less effective for long polypeptides.

8. Mass Spectrometry for Peptide Masses

• Measures peptide fragment masses, enabling protein identification against databases.

9. Protein Purification Methods

• Size: Gel filtration.

• Charge: Ion exchange chromatography.

• Affinity: Specific interactions with ligands.

• Solubility: Salting in/out.

10. Chromatography Overview

• Used to separate substances based on affinity to a solid:

  • Paper Chromatography: For small molecules.

  • Column Chromatography: For size/affinity-based separation.

11. Electrophoresis

• PAGE: Separates proteins by size and charge under an electric field.

• Western Blotting: Visualization of proteins.

12. Two-Dimensional Gel Electrophoresis

• Combines isoelectric focusing and mass separation for protein profiling.

13. Tertiary and Quaternary Protein Structures

• Tertiary structure: 3D shape from R-group interactions.

• Quaternary structure: Arrangement of multiple polypeptide subunits.

14. Important Concepts in Protein Structure

• Secondary Structure: Alpha-helices and beta-sheets stabilized by hydrogen bonds.

• Impact of residues: Proline disrupts helices; Glycine increases flexibility.

15. Quiz Questions

• Review questions on sequencing, purification, and structural elements for comprehensive understanding.