Recording-2025-03-06T20:29:40.920Z

Secondary Structure of Proteins

• Secondary structure refers to the localized folding patterns of a polypeptide chain due to hydrogen bonding.

  • Common types include alpha helices and beta sheets.

X-ray Crystallography

• X-ray crystallography is a technique used to determine protein structure through a five-step process:

  1. Protein Preparation: Must obtain a sufficient amount of pure protein.

  2. Crystal Formation: Incubate the pure protein in hundreds of conditions to encourage crystallization.

     • Crystals are compared to gummy bears, containing water.

  3. X-ray Exposure: The crystal is exposed to an X-ray beam, causing the rays to deflect off the electron cloud of the protein.

  4. Detection: Rays that bounce are detected, creating a diffraction pattern.

     • This pattern enhances certain signals while others are canceled out.

  5. Electron Density Map: Convert the diffraction pattern into an electron density map, allowing the fitting of amino acid sequences to determine the protein's structure.

Nuclear Magnetic Resonance (NMR) Spectroscopy

• NMR spectroscopy is a technique that analyzes protein structure in solution rather than crystals, offering a different perspective:

  • Complexity: More complicated than X-ray crystallography.

  • Size Limitations: Currently, NMR typically analyzes proteins up to 100 kilodal tons.

  • Information Provided: NMR provides distances between atomic nuclei, offering insights into the molecular environment and structural constraints within the protein.

  • Mechanism: Involves applying electromagnetic radiation to induce energy transitions in nuclei, aligning them with an external magnetic field. When they return to baseline, they emit signals that indicate distances between hydrogen atoms.

Comparing X-ray Crystallography and NMR

• X-ray crystallography provides electron density information and can analyze large complexes without size limitations. NMR provides distance measurements between atoms in solution, giving insight into the protein's folded structure.

• Importance of both methods:

  • Both methods can yield similar structures if the measurements are consistent despite different methodologies.

  • Large proteins often studied in fragments (domains) to overcome NMR limitations.

Visualization of Protein Structures

• Visualization techniques vary based on goals:

  • Ball and Stick Models: Displays every atom but can be cluttered.

  • Space-filling Models: Focus on protein surface, useful for understanding binding sites and active sites.

  • Artistic representations: Provide general shapes and secondary structures but lack precision.

General Principles of Protein Structure

• Understanding protein folding and interactions is guided by:

  • Side Chain Location:

    • Nonpolar residues predominantly found at the protein's core (interior).

    • Charged residues typically located on the surface (hydrophilic interactions).

    • Polar residues may be found both on the surface and inside, often involved in hydrogen bonding.

• Domain Structure:

  • Large proteins are often composed of independent domains, often containing at least two layers of secondary structure.

  • Binding sites for small molecules are usually situated between domains or subdomains.

Examples of Protein Motifs and Domains

• Beta-alpha-beta Motif: Folded structure combining beta sheets and alpha helices.

• Immunoglobulin Domain: Characteristic of antibodies. Contains multiple motifs working together.

• SRC Kinase: A specific domain found to transfer phosphate from ATP to tyrosine residues in other proteins, demonstrating functionality via binding grooves.

• General Protein Functionality: Binding sites within structural domain interface facilitate interactions with small molecule substrates, exemplified by enzymes like glycerol 3-phosphate.