Chapter 7 – Protein Function and Evolution

Overview

This chapter explores the biochemical principles underlying protein function and evolution, with a focus on analytical techniques, protein structure stabilization, and the molecular mechanisms of hemoglobin and myoglobin.

Protein Analysis Techniques

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gel electrophoresis is a widely used method for separating proteins and nucleic acids based on size and charge. Different types of PAGE provide information about protein structure and composition.

• Native Gel: Preserves the molecule's native structure; proteins migrate based on their charge and size.

• Denaturing Gel: Uses SDS (for proteins) or urea (for nucleic acids) to destroy native structure, allowing separation based solely on molecular weight.

• Reducing Gel: Disulfide bonds are broken by reducing agents (e.g., DTT, β-mercaptoethanol), enabling analysis of subunit composition.

• Non-Reducing Gel: Disulfide bonds are preserved, maintaining quaternary structure.

Key Chemicals:

• SDS (Sodium dodecyl sulfate): Anionic detergent that denatures proteins and imparts a uniform negative charge.

• Urea: Denaturant for nucleic acids and proteins (8–10 M).

• Guanidinium chloride: Strong denaturant (6–8 M), not suitable for electrophoresis due to charge and concentration.

• DTT and β-mercaptoethanol: Reducing agents for breaking disulfide bonds.

Principle: In SDS-PAGE, proteins are separated by molecular weight, as SDS masks intrinsic charge differences.

DNA and Protein Blotting Techniques

• Southern Blot: Detects specific DNA sequences using a labeled DNA probe.

• Northern Blot: Detects specific RNA sequences using a labeled DNA probe.

• Western Blot: Detects specific proteins using antibodies after separation by electrophoresis.

Applications: These techniques are essential for molecular biology research, diagnostics, and protein characterization.

Protein Structure Stabilization

Interactions Stabilizing Protein Structure

Proteins are stabilized by a variety of non-covalent interactions:

• Hydrogen Bonds: Form between polar groups, stabilizing secondary and tertiary structures.

• Salt Bridges: Electrostatic interactions between oppositely charged side chains.

• Hydrophobic Interactions: Nonpolar side chains aggregate to minimize contact with water, driving folding.

• Van der Waals Forces: Weak attractions between all atoms in close proximity.

Protein-Protein Interactions: Driven by weak interactions, these are crucial for cellular signaling and complex formation.

Antibodies and Immune Response

Antibody Structure and Function

Antibodies (immunoglobulins) are proteins produced by B-lymphocytes that recognize specific antigens.

• Antigen: Substance that elicits an immune response.

• Epitope: Specific region of the antigen recognized by the antibody.

• Antibody Structure: Composed of two heavy and two light chains, forming a Y-shaped molecule. The variable regions at the tips of the Y form the antigen-binding sites.

• Immunoglobulin Fold: A β-sandwich motif found in antibody domains.

• Complementarity-Determining Regions (CDRs): Hypervariable loops responsible for antigen specificity.

Binding Mechanism: Antibody-antigen interactions are mediated by shape and charge complementarity, involving hydrogen bonds, hydrophobic interactions, and van der Waals forces.

Applications: High specificity of antibodies is exploited in diagnostics and targeted drug delivery.

Hemoglobin and Myoglobin: Structure and Function

Overview and Comparison

Hemoglobin and myoglobin are heme-containing proteins essential for oxygen transport and storage.

• Myoglobin: Monomeric protein found in muscle tissue; stores and releases oxygen.

• Hemoglobin: Tetrameric protein in red blood cells; transports oxygen from lungs to tissues and returns CO2 for exhalation.

• Heme Group: Iron-containing porphyrin ring; Fe2+ binds oxygen.

• Apoprotein: Protein without heme.

• Holoprotein: Protein with heme.

Oxygen Binding: O2 binds to Fe2+ in the heme, coordinated by four nitrogen atoms and a proximal histidine residue.

Oxygen Binding Curves

Oxygen binding to myoglobin and hemoglobin can be described mathematically and graphically.

• Myoglobin: Exhibits a hyperbolic binding curve, indicating non-cooperative binding.

• Hemoglobin: Exhibits a sigmoidal binding curve, indicating cooperative binding.

Fractional Saturation Equation:

• P50: Partial pressure of O2 at which the protein is half-saturated; lower P50 indicates higher affinity.

Allosteric Regulation of Hemoglobin

Allostery and Cooperativity

Hemoglobin's oxygen binding is regulated by allosteric transitions between two states:

• T (Tense) State: Lower affinity for O2; stabilized by salt bridges and H-bonds.

• R (Relaxed) State: Higher affinity for O2; stabilized by O2 binding.

Models of Allosteric Transition:

• KNF Model (Sequential): O2 binding induces conformational change in one subunit, facilitating transition in adjacent subunits.

• MWC Model (Concerted): Hemoglobin exists in equilibrium between T and R states; O2 binding shifts equilibrium toward R state.

Cooperativity: Binding of O2 to one heme increases affinity at other hemes (positive homotropic allostery).

Allosteric Effectors

Hemoglobin's affinity for oxygen is modulated by several effectors:

• Homotropic Effectors: O2 itself; binding increases further O2 affinity.

• Heterotropic Effectors: H+ (pH), CO2, and 2,3-bisphosphoglycerate (BPG); binding decreases O2 affinity (negative allostery).

Bohr Effect: Lower pH (higher H+) stabilizes T state, promoting O2 release in tissues.

CO2: Forms carbamate with hemoglobin, releasing H+ and contributing to Bohr effect.

BPG: Binds in the central cleft of T state, stabilizing it and promoting O2 release.

Fetal Hemoglobin: Reduced BPG binding increases O2 affinity, facilitating maternal-fetal O2 transfer.

Protein Evolution and Hemoglobinopathies

Evolutionary Relationships

Protein evolution is traced by comparing amino acid sequences and mutation rates in homologous proteins.

• Gene Duplication: Leads to new protein functions and evolutionary branches.

• Globin Family: Conserved fold and critical residues across species.

Hemoglobinopathies

Genetic mutations in hemoglobin can lead to inherited diseases.

• Sickle-Cell Disease: Caused by a single amino acid substitution (Glu to Val) in β-globin, resulting in abnormal erythrocyte shape, blockage of capillaries, and anemia.

• Heterozygote Advantage: Carriers are resistant to malaria under oxygen stress.

Summary Table: Hemoglobin vs. Myoglobin

Key Equations

• : Partial pressure of O2 at half saturation

Additional info: Some context and explanations have been expanded for clarity and completeness, including definitions, mechanisms, and applications relevant to biochemistry students.