LECTURE 11: Protein sequencing and evolution/ Secondary structure

Chromatography Techniques

• Ion Exchange Chromatography

  • Separates based on the charge density on the surface of the protein.

  • Cation Exchange Chromatography: Column is negatively charged, binding positively charged groups on the protein.

  • If protein binds too strongly, adjust pH or increase salt concentration to elute the protein.

• Gel Filtration Chromatography

  • Separates based on size. Larger molecules elute first; smaller molecules elute later.

• Hydrophobic Interaction Chromatography

  • Separates based on the hydrophobicity of the protein surface.

• Affinity Chromatography

  • Separates based on specific interactions between proteins and other molecules.

  • Example: Purifying hexokinase using ATP or glucose as a ligand for binding.

Gel Electrophoresis

• Gel Electrophoresis: Used to separate proteins or nucleic acids based on size.

  • Can be done with or without SDS (Sodium Dodecyl Sulfate).

  • SDS: Anionic detergent that imparts a negative charge and denatures proteins by disrupting hydrophobic interactions.

  • Beta-Mercaptoethanol: Reducing agent used to break disulfide bonds between cysteine residues in proteins.

• Isoelectric Focusing

  • A technique using a pH gradient to separate proteins based on their isoelectric point (pI).

  • Isoelectric point is where a molecule has no net electrical charge and does not move in an electric field.

Protein Sequencing

• General Process: Sequentially remove and identify amino acids to determine the protein sequence.

  • First amino acid is modified, and the bond is broken, allowing for identification.

  • Successive cycles refine the sequence through this method.

• Peptide Sequencing Challenges: Involves chopping proteins into smaller peptides for analysis, sometimes using specific enzymes like trypsin.

  • Overlapping sequences from different cleavages can help reconstruct the original protein sequence.

Molecular Evolution and Protein Variability

• Comparison of protein sequences across species shows:

  • Invariant Residues: Amino acids that remain unchanged across species.

  • Conservative Substitutions: Similar amino acids substituting for each other, maintaining similar properties.

  • Hypervariable Residues: Positions in proteins where any amino acid can be present, indicating flexibility in the protein structure.

• Evolution influences protein sequences through mutations, with some mutations promoting fitness, others leading to a loss of function.

Protein Structure Levels

• Primary Structure: Sequence of amino acids in a polypeptide chain.

• Secondary Structure: Local folding/spatial arrangement (alpha helices and beta sheets).

• Tertiary Structure: Overall three-dimensional shape of a single polypeptide, including side chains.

• Quaternary Structure: Complex of multiple polypeptide subunits interacting together.

Forces Stabilizing Protein Structure

• Primary Structure: Stabilized by covalent bonds.

• Secondary, Tertiary, and Quaternary Structures: Stabilized by non-covalent interactions (hydrophobic interactions, hydrogen bonds, ionic bonds).

  • Disulfide bonds may also play a role in stabilization, especially in extracellular proteins.

Conclusion and Next Steps

• Upcoming topics include the specific functions and folding of proteins, focusing on fibrous proteins like collagen and keratin, and globular proteins including myoglobin and hemoglobin.

• Understanding protein structure aids in comprehending biological functions.