m4p5

Module 4 Overview

  • Basic protein purification techniques are foundational in modern biochemistry laboratories.

    • The primary methods include:

      • Chromatography

      • Electrophoresis

  • The module examines three main approaches biochemists utilize to determine protein three-dimensional structures:

    • X-ray Crystallography

    • Nuclear Magnetic Resonance (NMR) Spectroscopy

    • Cryo-Electron Microscopy (Cryo-EM)

  • Proteins are crucial for cellular functions, performing roles such as:

    • Catalyzing biochemical reactions

    • Organizing cell structures

    • Transporting biomolecules

    • Transducing cellular signals

    • Managing genetic information

  • The exploration of protein structure leads to a discussion of the major functional classes of proteins and how these structures relate to the function of hemoglobin.

Module 4 Lecture Topics and Textbook Readings

Topic

Lecture Title

Textbook Readings

1

Protein Purification; Chromatography

Chapter 5.1

2

Protein Purification; Electrophoresis

Chapter 5.1

3

Protein Structure Methods

Chapters 5.2, 5.3

4

Major Protein Classes

Chapter 6.1

5

Hemoglobin: Structure and Function

Chapter 6.2

6

Oxygen Binding to Hemoglobin

Chapter 6.2

Hemoglobin: Structure and Function

  • Fresh meat exhibits a red color due to the presence of ferrous ion (Fe2+) in hemoglobin.

    • When meat spoils, the iron oxidizes to ferric ion (Fe3+), resulting in a brown color.

Key Concepts: Oxygen Transport

  • Myoglobin: Monomeric protein functioning as an O2 storage protein in tissues.

  • Hemoglobin: Tetrameric protein responsible for O2 transport from lungs to tissues.

  • O2 Binding Mechanism:

    • The binding of O2 to hemoglobin invokes conformational changes transitioning from the T (tense) state to the R (relaxed) state.

    • These changes influence the affinity of other subunits for O2 binding.

  • The binding process involves:

    • Coordination of O2 to the iron atom in the heme group.

    • Movement of the F helix corresponding to the positioning of Fe within the heme structure.

Oxygen Transporting Proteins

  • Both myoglobin and hemoglobin are heme-utilizing oxygen-binding proteins composed of:

    • Eight α helices per subunit

    • A heme molecule bound to each subunit.

  • Hemoglobin is classified as a heterotetramer.

  • Structural Comparison:

    • The globin fold of myoglobin is structurally similar to both types of hemoglobin subunits despite low amino acid sequence similarity between myoglobin and the hemoglobin α or β subunits.

Molecular Mechanism of Oxygen Transport

  • In both myoglobin and hemoglobin:

    • A proximal histidine residue coordinates with Fe2+.

    • A distal histidine forms a hydrogen bond with O2, stabilizing its interaction with the heme group.

  • In the absence of O2:

    • Fe2+ remains out of the plane of the porphyrin ring due to its larger size.

    • Upon O2 binding, electron sharing reduces the ionic radius of Fe2+, allowing it to move into the porphyrin plane.

    • This movement induces a shift in the F-helix of the polypeptide chain.

Structural Changes Upon Oxygen Binding

  • The structures of oxyhemoglobin and deoxyhemoglobin show:

    • A displacement of 0.6 Å in the position of His F8 results in a corresponding 1 Å tilt in the F-helix.

  • These molecular alterations emphasize the importance of protein conformational dynamics in the process of oxygen transport.