Membrane Proteins

Recombinant Expression Systems:

When a natural source is unavailable, researchers must synthesise the protein. Each system has distinct advantages and limitations.

  • Bacteria (E. coli): While cheap and simple, they are often "lousy" at making mammalian membrane proteins.

    • Codon Bias: Humans and bacteria use the 64 available codons differently. For example, there are six codons for Leucine; a bacterium might lack the specific tRNA for a Leucine codon that is common in humans.

    • Post-translational Modifications: Bacteria do not glycosylate or phosphorylate proteins correctly and often fail to form necessary disulfide bonds.

  • Insect Cell System (Baculovirus): This system uses a cell line derived from the Army Fall Worm Moth.

    • Mechanism: Researchers take the promoter for a protein called polyhedrin (which naturally crystallises in the caterpillar's gut) and place it upstream of the gene of interest on a plasmid.

    • Infection: This modified virus infects the moth cells, forcing them to produce large quantities of the target protein.

    • Benefit: As eukaryotes, insect cells correctly perform phosphorylation, glycosylation, and disulfide bond formation.

  • Cell-Free Systems: These utilise isolated ribosomes and rough endoplasmic reticulum without the rest of the cell. They are extremely expensive and typically reserved for proteins that are toxic to living cells.

Detergent Solubilisation and the CMC:

Detergents are used to "rip" proteins out of the membrane by shifting the balance between hydrophobic and hydrophilic properties.

  • Chemical Structure: Lipids generally have two hydrophobic chains, making them water-insoluble (forming bilayers). Detergents typically have only one hydrophobic tail and one hydrophilic head, making them 5 to 6 orders of magnitude more water-soluble than lipids.

  • Critical Micelle Concentration (CMC): This is the threshold where detergent monomers spontaneously aggregate into micelles.

    • Adding detergent to a clear solution causes it to suddenly become turbid at the CMC as micelles begin to disrupt light transmission.

    • Once micelles form, they surround the protein and lipids with a hydrophilic "outer shell," bringing the hydrophobic protein into solution.

  • Detergent Selection:

    • SDS: A "gold star" for solubilization because it dissolves almost anything, but it is often avoided because it denatures (unfolds) the protein.

    • DDM and DM: Preferred detergents that often solubilise a large amount of protein without destroying its structure.

    • Length Mismatch: Because membranes vary in thickness based on their fatty acid chain length, researchers must match the length of the detergent (e.g., 8, 10, or 12 carbons) to the size of the protein's transmembrane domain.

Purification and Reconstitution:

Purifying membrane proteins requires keeping them in a detergent environment to prevent aggregation and precipitation.

  • Chromatography Hurdles:

    • Affinity: The detergent shell can physically obscure the "tag" (like a His-tag) used for purification.

    • Size Exclusion: Detergent adds a "shell" that makes the protein appear significantly larger than its actual molecular weight.

  • Reconstitution: Pure proteins in detergent are often inactive because they lack a bilayer for directionality (e.g., a transporter cannot transport if there is no physical barrier to cross).

  • The Annular Ring: Proteins have "preferred" lipids they want to be embedded in, known as the annular ring. Using the wrong lipids during reconstitution can result in a non-functional protein.

  • Cookie-Cutter Polymers (SMA): A specialized polymer (Styrene Maleic Acid) can "hole punch" a section of the native membrane. This keeps the protein inside a small "cookie" of its original lipids, maintaining its native environment while remaining water-soluble.

Structural Biology Challenges:

Membrane proteins are notoriously difficult to study due to several factors discussed by Professor Kerr:

  • Genome Dedication: About 25% of the human genome is dedicated to encoding membrane proteins, and roughly 50% of all drugs target them.

  • Resolution: In structural biology, resolution is a measure of how well the position of each atom is known.

    • Low Resolution (e.g., 5 Å): Appears as a "meshwork" where amino acids look like undifferentiated "blobs".

    • High Resolution (e.g., 1.5–2 Å): Allows researchers to clearly trace rings and side chains, such as tryptophan.

  • Crystal Growth: Unlike soluble proteins, membrane proteins are hard to grow into three-dimensional crystals because they naturally exist in a two-dimensional environment (the membrane).

The Electron Microscopy (EM) Revolution:

  • Single Particle Imaging: Instead of needing 2D crystals, researchers take millions of images of individual protein particles.

  • Image Stabilisation: Modern software can "stabilise" images of moving proteins—similar to the technology in a smartphone camera—to produce a static, high-resolution average.

  • Vitreous Ice: Samples are frozen rapidly in liquid ethane (cooled by liquid nitrogen), creating "vitreous ice" that is invisible to electrons, allowing the protein structure to be viewed without interference.

The Discovery of Aquaporins:

The "Contaminant":

While studying the Rhesus antigen (a 30-32 kDa protein) in red blood cells, Agre's team noticed a persistent 28 kDa "contaminant" on their protein gels.

  • This protein had been overlooked for decades because it does not stain well with Coomassie Blue; it was only visible using silver staining.

  • It is incredibly abundant, with roughly 200,000 copies per red blood cell.

The Functional Test:

Agre hypothesised this protein was a water channel. He tested this using Xenopus laevis (African clawed frog) oocytes, which are naturally impermeable to water:

  1. Injection: They injected the oocytes with the messenger RNA for the 28 kDa protein.

  2. Osmotic Shock: When placed in water, the injected cells swelled up and burst "like a ballet balloon," while the control cells remained unchanged.

  3. Result: This proved the protein was a water channel, later named Aquaporin.

Aquaporin Structure and Mechanism:

Aquaporins are highly specific "pores" that allow water through while strictly excluding ions and protons.

The "NPA" Motif:

Aquaporins contain a highly conserved Asn-Pro-Ala (NPA) motif. This motif is found in organisms ranging from E. coli to elephants, indicating its critical evolutionary role.

Excluding Protons (The Grotthuss Mechanism):

Normally, protons can "hop" across a line of water molecules if their dipoles are aligned. Aquaporin prevents this "proton wire" through its unique architecture:

  • Hourglass Shape: The channel is too narrow (less than 2.5 Å) to let through solvated ions.

  • Dipole Reorientation: In the centre of the pore, the water molecules are forced to bind to the asparagine residues of the NPA motif.

  • Breaking the Chain: This interaction breaks the continuous chain of hydrogen bonds between water molecules, preventing protons from hopping through while allowing individual water molecules to pass.