17/4 Lecture Notes on Proteins and Membrane Transport

Introduction to Proteins

• Today's focus is on proteins, particularly an ADP ATP transporter.

• An average human cell can express around 20,000 different proteins, with about 10,000 present depending on cell type.

• Approximately 2,000 proteins are associated with membranes, facilitating communication outside the cell or organelle.

• Organelles separate important pathways and processes within the cell.

• Membrane proteins:

  • Receptors: Recognize signals (e.g., hormones like glucagon receptor).

  • Channels: Allow ions to pass in and out.

  • Transporters: Require some input for controlled movement of molecules.

Levels of Protein Structure

• Primary Structure: The amino acid sequence.

• Secondary Structure: Organization of amino acids into structures like alpha helices and beta sheets. (chain organisation, domains)

• Tertiary Structure: How secondary structure elements combine to form a functional protein. (domain organisation, ordered peptide)

• Quaternary Structure: Association of multiple protein subunits for function. (interaction of multiple structured peptides)

  • Example: Aldolase, which functions as a tetramer (four subunits).

Stabilization of Protein Structures

• Primary Structure:

  • Amino acids are linked by peptide bonds (carbon and nitrogen linkage).

  • Peptide bond is stable and has a planar arrangement, important for protein folding.

  • Condensation reaction: water is released.

  • Peptide bonds broken by proteases, not by detergents used in aldolase prac.

  • Acid hydrolysis required to break these bonds under harsh conditions

Amino Acids and Their Characteristics

• 20 different amino acids with varying R groups.

• Polar amino acids: hydrophilic and interact with water.

  • Polar uncharged.

  • Polar charged: Positively and negatively charged R groups.

• Hydrophobic non polar amino acids: avoid interaction with water.

  • The arrangement of these amino acids determines protein folding.

Weak Interactions Driving Protein Folding

• Protein folding is driven by a combination of weak interactions:

  • Hydrogen bonds: interaction between electronegative atom (oxygen) and electropositive hydrogen.

  • Electrostatic interactions: interactions between oppositely charged amino acid side chains.

  • Van der Waals forces: temporal fluctuations in electron positions around atoms.

  • Hydrophobic effect: hydrophobic amino acids shield themselves from water and are buried inside the protein. (fats)

Secondary Structures

• Alpha Helices:

  • Hydrogen bonding between backbone residues.

  • Side chains project radially from the helix, not involved in stabilisation of structure.

  • Held together by hydrogen bonds between carbonyl and $NH$ groups.

  • One turn of the alpha helix requires $3.6$ residues.

• Beta Sheets:

  • Composed of beta strands. Strands are $5$ to $10$ residues long.

  • Hydrogen bonding between peptide backbones.

  • R groups project differently depending on whether it's a parallel or anti-parallel beta sheet.

• Loops:

  • Connect secondary structures.

  • Often have important functional roles

  • Cytochrome C Oxidase Example

    • Membrane protein with helices situated in the lipid membrane.

    • Loops and functional groups in the aqueous region.

    • Hydrophobic amino acids are associated with helices that interact with the hydrophobic lipid regions.

Tertiary Structure

• Arrangement of secondary structure elements.

• Driven by side chain interactions.

• Conserved amino acids in regions important for function, especially in enzymes.

• Proteins with almost no primary amino acid level sequence identity can have identical folds but not necessarily identical functions.

• Examples of interactions: Hydrophobic interactions, hydrogen bonding, and ionic interactions.

• Disulfide bridges: Strong bonds formed via oxidation, often associated with extracellular proteins in harsh environments; broken by reducing agents

• Energetically stable molecule arranged to satisfy interactions.

Structural Motifs

• Proteins grouped by how structures come together (e.g., helix-loop-helix).

  • interacting helices are arranged so that side-chains of one helix slot into the groove of a neighbour

• Beta-barrel structures:

  • Common in membrane proteins and bacterial toxins.

  • Can form pores in cells.

Quaternary Structure

• How tertiary structure units come together.

• Homo-oligomers: Multiple subunits of the same protein (dimers, trimers, tetramers).

• Hetero-oligomers: Multiple subunits of different proteins (e.g., F1ATPase).

Function of Membrane Proteins

• Capacity to perceive signals from outside the cell.

• Translate signals to change pathways inside the cell.

• Transporters facilitate movement of molecules.

ATP ADP Translocase

• Crucial protein for ATP production.

• We only have around $250$ grams of ATP in our body, and so what we need to do is we need to generate around about body weight in ATP every day to survive.

• Most ATP production occurs in the mitochondria (some in cytosol).

• ATP ADP Translocase Abundant protein (around $10$% of inner mitochondrial membrane protein).

• Facilitates stoichiometric (one-to-one) transport of ADP into the mitochondrial matrix and ATP out.

• Electrogenic transport: Influenced by charge difference across the membrane.

  • Intermembrane space: positively charged due to protons.

  • Matrix: negatively charged.

  • Favors ATP transport out (more negatively charged due to additional phosphate).

  • Phosphate is also transported in, important for ADP to ATP synthesis.

Structure of ATP ADP Translocase

• Six transmembrane helices (H1-H6) that embed in the membrane.

• Hydrophobic amino acids align with the lipid part of the membrane, while hydrophilic amino acids enable association with ATP and ADP.

• Two different conformations:

  • Open to the mitochondrial matrix to accept ATP binding.

  • Open to the intermembrane space to accept ADP binding.

• Cavity lining (hollow space/pockets) is hydrophilic.

• Lipid lining residues are hydrophobic.

• Charged residues (lysine, arginine) at the entrance of the cavity bind ATP （start bringing atp into cavity of protein).

• Aromatic residues (tyrosine) direct the sugar region.

• ATP binding forces a conformational change:

  • Conformational change releases ATP.

  • Transporter then accepts ADP for transport back across.

Role of Cardiolipins

• Cardiolipins are crucial for the activity of the protein.

• Disorders affecting cardiolipin production lead to muscle wastage and low energy production.

• Cardiolipins stabilize the structure in the membrane and facilitate conformational change.

• Prevent misfolding or aggregation of the protein.

Additional points:

• ATP, ADP are polar molecules, so they need a transporter to go across, and that enables us to control the transport of these really important molecules.

• Lipid membranes have hydrophobic regions.

Key points to review for your exam:

1. Protein Structure Levels: Primary, secondary, tertiary, and quaternary structures, and what each entails.

2. Stabilization of Protein Structures: Focus on peptide bonds, their characteristics, and how they're broken.

3. Amino Acids: The different types (polar, hydrophobic) and their roles in protein folding.

4. Weak Interactions: Hydrogen bonds, electrostatic interactions, Van der Waals forces, and the hydrophobic effect.

5. Secondary Structures: Alpha helices, beta sheets, and loops—their composition and importance.

6. Cytochrome C Oxidase: Understand its structure as a membrane protein and the arrangement of amino acids.

7. Tertiary Structure: How secondary structures arrange and the types of interactions involved. Pay special attention to disulfide bridges