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.

Analytical Techniques for Protein and Nucleic Acid Study

Polyacrylamide Gel Electrophoresis (PAGE)

Polyacrylamide gel electrophoresis is a fundamental method for separating proteins and nucleic acids based on size and charge. Different types of PAGE provide insights into protein structure and composition.

• Native Gel: Preserves the native structure of molecules, allowing analysis of protein complexes and conformations.

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

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

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

• Note: Guanidinium chloride is not used as a denaturant in electrophoresis due to its charge and high concentration requirements.

Key Formula:

Blotting Techniques

Blotting methods are used to detect specific biomolecules after electrophoresis.

• 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.

Example: Western blotting can identify the presence of a phosphorylated protein in a complex mixture.

Protein Structure Stabilization and Interactions

Interactions Stabilizing Protein Structure

Proteins are stabilized by several types of non-covalent interactions:

• Hydrogen Bonds: Form between polar groups, contributing to secondary and tertiary structure.

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

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

Weak Interactions: These also drive protein-protein interactions, essential for complex formation and signaling.

Antibody Structure and Function

Immunoglobulin Structure

Antibodies (immunoglobulins) are proteins that recognize specific antigens via their variable regions.

• Epitope: The specific part of an antigen recognized by an antibody.

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

• Hypervariable Regions (CDRs): Complementarity-determining regions at the ends of variable domains, responsible for antigen specificity.

Antibody-Antigen Binding: Mediated by shape and charge complementarity, involving hydrogen bonds, van der Waals forces, and hydrophobic interactions.

Application: High specificity of antibodies is exploited in targeted drug delivery.

Hemoglobin and Myoglobin: Structure and Function

Overview and Comparison

Hemoglobin and myoglobin are oxygen-binding proteins with distinct roles and structures.

• Myoglobin: Monomeric heme protein, facilitates oxygen storage and delivery in muscle tissue.

• Hemoglobin: Tetrameric heme protein, transports oxygen from lungs to tissues and returns CO2 for exhalation.

• Heme Group: Iron protoporphyrin IX; Fe2+ binds O2 and is coordinated by histidine residues.

• Apoprotein: Protein without heme; Holoprotein: Protein with heme.

Example: Deep-diving mammals have higher myoglobin concentrations for increased oxygen storage.

Oxygen Binding and Affinity

Oxygen binding to myoglobin and hemoglobin can be described quantitatively.

• Fractional Saturation (): Proportion of oxygen-binding sites occupied.

• P50: Partial pressure of oxygen at half saturation; lower P50 indicates higher affinity.

Key Equations:

Oxygen Binding Curves

Myoglobin exhibits a hyperbolic binding curve, while hemoglobin shows a sigmoidal curve due to cooperative binding.

• Hyperbolic Curve: Indicates non-cooperative binding (myoglobin).

• Sigmoidal Curve: Indicates cooperative binding (hemoglobin), allowing efficient oxygen loading and unloading.

Allosteric Regulation of Hemoglobin

Allostery and Cooperativity

Allosteric regulation involves conformational changes in a protein upon ligand binding, affecting activity at other sites.

• Homotropic Allostery: Effector is the substrate itself (e.g., O2 for hemoglobin).

• Heterotropic Allostery: Effector is a different molecule (e.g., H+, CO2, BPG for hemoglobin).

• Positive Allostery: Effector increases activity or affinity.

• Negative Allostery: Effector decreases activity or affinity.

Key Models:

• KNF Model: Sequential model; ligand binding induces conformational change in one subunit, facilitating changes in adjacent subunits.

• MWC Model: Concerted model; protein exists in two states (Tense, T; Relaxed, R), and ligand binding shifts equilibrium toward R state.

T and R States of Hemoglobin

Hemoglobin transitions between T (low affinity) and R (high affinity) states upon oxygen binding.

• T State: Stabilized by more noncovalent intersubunit interactions (salt bridges, H-bonds).

• R State: Fewer intersubunit interactions, higher oxygen affinity.

• Transition: Oxygen binding pulls Fe2+ into the heme plane, triggering conformational changes.

Allosteric Effectors of Hemoglobin

Several molecules modulate hemoglobin's oxygen affinity by stabilizing either the T or R state.

• O2: Positive homotropic effector; binding increases affinity for additional O2.

• H+ (Bohr Effect): Negative heterotropic effector; increased H+ (lower pH) stabilizes T state, promoting O2 release.

• CO2: Negative heterotropic effector; binds to amino groups, forming carbamate and releasing H+.

• 2,3-Bisphosphoglycerate (BPG): Negative heterotropic effector; binds in the central cleft of T state, reducing O2 affinity.

Example: Fetal hemoglobin has reduced BPG binding, resulting in higher O2 affinity for efficient oxygen transfer from mother to fetus.

Protein Evolution and Hemoglobinopathies

Evolutionary Relationships

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

• Gene Duplication: Leads to new protein families; more recent duplications yield more closely related proteins.

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

Hemoglobinopathies

Genetic mutations in hemoglobin can lead to diseases such as sickle-cell anemia.

• 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 asymptomatic except under oxygen stress and have increased resistance to malaria.

Summary Table: Hemoglobin vs. Myoglobin

Additional info: Some context and definitions have been expanded for clarity and completeness.