U02 p3

Unit Overview

  • Course: BIOL 331 – Molecular Cell Biology

  • Source Material: Molecular Biology of the Cell, 6th edition by Alberts et al.

  • Content Focus: Chapter 10, pages 603 - 633.

Membrane Structure and Function

Key Topics Covered

  • Membrane Proteins: Solubilization and purification techniques.

  • Diffusion in Membranes: Movement and confinement of proteins and lipids.

  • Role of Cytoskeleton: Structural support and diffusion restrictions.

  • Membrane Bending Proteins: Influence on membrane shape and conformation.

Membrane Proteins Can Be Solubilized and Purified in Detergents

  • Integral Membrane Proteins:

    • Solubilization requires disrupting hydrophobic interactions.

    • Detergents are the primary tools used.

  • Detergent Properties:

    • Small, amphiphilic molecules.

    • Exist in various shapes, more soluble in water than lipids.

    • Head groups can be ionically charged (e.g., SDS) or uncharged (e.g., Triton X-100).

Techniques for Solubilizing Membrane Proteins

  • Critical Micellar Concentration (CMC):

    • The concentration at which detergents form micelles.

    • Above this threshold, the behavior of detergent molecules changes.

    • CMC affected by temperature, pH, and ionic strength.

  • Mild vs. Strong Detergents:

    • Mild detergents (non-ionic) can solubilize proteins without denaturing them.

    • Strong detergents (ionic) completely denature proteins.

Methodology for Studying Membrane Proteins

  • Solubilization with mild detergents, followed by:

    • Lipid concentration reduction, encouraging membrane proteins to self-assemble into liposomes.

    • Useful for biochemical characterization of ion transporters, signaling receptors, etc.

Reconstituting Membrane Proteins in Nanodiscs

  • Nanodiscs: Small patches of membrane with a protein belt made of high-density lipoproteins.

    • Maintain solubility and mimic natural environments.

    • Allow for structural analysis without crystallization.

Protein Diffusion in Membranes

  • Diffusion Mechanics:

    • Proteins can diffuse in the lipid bilayer.

    • FRAP (Fluorescence Recovery After Photobleaching) technique measures diffusion rates.

    • Proteins tagged with fluorescent ligands or GFP for tracking.

Confinement of Proteins and Lipids

  • Apical-Basal Polarity:

    • Epidermal cells exhibit polarity with distinct protein localization (apical vs. basal regions).

    • Tight junctions serve as barriers to lateral diffusion.

  • Alternative Restriction Mechanisms:

    • Self-assembly into aggregates or tethering interactions (both intra- and extracellular).

Role of Cytoskeleton in Protein Diffusion

  • Cytoskeletal connections restrict lateral movement of membrane proteins.

  • Example: The biconcave shape of erythrocytes is maintained by spectrin interactions with the plasma membrane.

  • Other cell types may have more complex cytoskeletal arrangements affecting their membrane structures.

Membrane Bending Proteins

  • Variety in membrane shapes and structures dictated by membrane-bending proteins.

  • These proteins insert between phospholipids and can force membranes into desired configurations through their polar head groups.