Insulin & Aquaporin Case Study

Take-Home Point

  • Life works because of proteins; when proteins do not function correctly, disease often occurs.

  • Protein function is dictated by protein structure.

  • Protein structure can be explained through basic chemistry concepts, including hydrogen bonding learned in general chemistry.

Learning Outcomes

  1. Define the four levels of protein structure:

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

    • Secondary Structure: Local folding of the polypeptide into alpha-helices and beta-sheets due to hydrogen bonding patterns.

    • Tertiary Structure: Overall 3D shape of a polypeptide, stabilized by various interactions.

    • Quaternary Structure: Assembly of multiple polypeptide chains into a functional protein complex.

    • Identify the chemical bonds contributing to each level:

      • Primary: Peptide bonds.

      • Secondary: Hydrogen bonds.

      • Tertiary: Disulfide bonds, ionic bonds, hydrogen bonds, hydrophobic interactions, van der Waals forces.

      • Quaternary: Same as tertiary, between different polypeptide chains.

  2. Explain the contributions of enthalpy (ΔH) and entropy (ΔS) to protein folding:

    • Enthalpy reflects the heat of formation associated with bond formation and breaking.

    • Entropy is a measure of disorder; favorable folding increases overall molecular organization.

  3. Identify the most important commonality among all non-covalent interactions:

    • They all involve transient forces that play roles in biochemical interactions and conformational stability.

  4. Explain why the energies of formation are different for different non-covalent interactions:

    • Variations in charge, bond energies, and molecular configurations influence interaction energies.

  5. Interpret the formula:


    • ext{ΔG} = ΔH - TΔS
      to explain the role of enthalpy and entropy in determining the spontaneity of a reaction (Gibb’s free energy).

    • Apply this formula to the context of protein folding.

  6. Predict the impact of changes in amino acid substitutions:

    • Amino acid substitutions can alter the folding, stability, and functionality of cytoplasmic proteins.

  7. Describe amino acid distribution in soluble and transmembrane proteins:

    • Compare and contrast the location and distribution of hydrophobic, polar neutral, and polar charged amino acids in both types of proteins.

  8. Examine amino acid side chains:

    • Determine the predominant non-covalent interactions that occur between two amino acid side chains.

  9. Explain aquaporin channel selectivity:

    • Aquaporin channels maintain selectivity for water molecules and exclude protons and other ions (e.g., Na+, Cl-) through specific structural features and interactions.

Central Dogma

  • Peptide bonds are formed during translation by the ribosome. It is catalyzed by rRNA, which is referred to as a ribozyme.

  • Synthesis of mRNA Process:

    • mRNA is synthesized from DNA in the nucleus then transported to the cytoplasm where it is translated into polypeptides.

Levels of Protein Structure

  • Reference: Khan Academy on Orders of Protein Structure

  • Protein Structure Levels:

    • Primary: Peptide bonds formed between amino acids.

    • Secondary: Hydrogen bonds formed between the backbone of the polypeptide chain.

    • Tertiary: Various bonds (e.g., ionic, hydrogen, hydrophobic interactions) formed between side chains.

    • Quaternary: Bonds formed between polypeptide subunits.

Protein Folding

  • Influence of the Hydrophobic Effect:

    • The hydrophobic effect drives nonpolar amino acid side chains to the interior of the protein, affecting how proteins fold in aqueous environments.

Insulin Structure

  • Insulin Composition:

    • Insulin has A and B chains connected by disulfide bonds.

  • Amino Acid Distribution in Insulin (PDB entry 1TRZ):

    • Highlights sections of the A-chain (hydrophilic/hydrophobic regions) and B-chain showing the distribution of charged surfaces and disulfide linkages.

Non-Covalent Interactions

  • Role in Protein Folding:

    • Types of non-covalent interactions include:

    1. Ion-Ion Interactions:

      • Involve full charges; generally strong and permanent.

    2. Hydrogen Bonds:

      • Interactions with full or partial charges; strong if permanent.

    3. Van der Waals:

      • Temporary interactions; weakest of all.

  • Ion-Dipole Interactions:

    • Involves interaction between charged molecules and polar molecules.

Impact of Thermodynamics on Protein Folding

  • 2nd Law of Thermodynamics:

    • The total entropy of a system and its surroundings always increases in a spontaneous process.

  • Gibbs Free Energy:


    1. ext{ΔG} = ΔH - TΔS

    2. Each state can be evaluated on whether it is spontaneous or not based on temperature vs ΔH and ΔS relations.

Protein Folding vs Denaturation

  • Free energy differences between folded and unfolded states can be modest, resulting from a few non-covalent bonds.

  • Conditions like temperature and pH can impact protein folding and stability.

Mutation in Insulin

  • Considerations affecting functionality include amino acid polarity, ionization state at pH 7.4, and tertiary/quaternary interactions.

  • Mutations can affect how a protein folds and its resultant activity.

Aquaporin Function

  • Aquaporins in Water Transport:

    • Facilitate rapid water transport across membranes; significant in kidney function and plant transpiration.

  • Table of Mammalian Aquaporins and Associated Diseases:

    • Provides features of various aquaporins, their selectivity for water or other substances, localization in tissues, and diseases associated with mutations.

Membrane Structure and Protein Interaction

  • Composition of Membrane Molecules:

    • Phospholipid bilayers with polar head groups and nonpolar tails; interactions define membrane properties.

  • Aquaporin Structure:

    • The aquaporin structure is critical for channel function and permeation characteristics.

Mechanisms of Ion Flux and Selectivity

  • Selectivity Mechanisms in Aquaporins:

    • Facilitates water passage while repelling protons and other ions through specific structural features.

    • Grotthuss Mechanism: Describes how water molecules can transport protons through hydrogen bonds in a chain reaction but is prevented in aquaporins via architectural adaptations.

Aquaporin Channel Properties

  • The NPA Motif (Asparagine-Proline-Alanine) plays a crucial role in maintaining selectivity for water molecules and preventing proton conduction.

  • Analogy of Functionality:

    • Hula hoop analogy is used to describe how disruption of hydrogen bonding allows for selective water passage.

Learning Outcomes Related to Water and Proteins

  1. Identify phospholipid components and their assembly into biological membranes.

  2. Define various non-covalent interactions and recognize them in biomolecules.

  3. Explain influence of entropy, van der Waals interactions on the hydrophobic effect, etc.

  4. Classify amino acids based on polarity.

  5. Use pKa to determine predominant ionization states at different pH levels.

  6. Apply the Henderson-Hasselbach equation for calculating the charge on amino acids.

  7. Draw peptides with defined characteristics.

  8. Explain hydrogen bonding related to alpha helical structure.

  9. Compare modeling techniques in depicting alpha helical structure.