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Membrane proteins
Uni of Notts, Structure, function, & analysis of Proteins, year 2, topics 14-15
| Term | Definition |
|---|---|
| Peripheral & anchor proteins | Peripheral proteins associate non-covalently with membranes; anchor proteins are covalently lipid-linked. Both have hydrophobic cores & hydrophilic shells |
| Why some membrane proteins have hydrophilic cores | To form channels/pores for transport of polar molecules across membranes |
| Why polar residues are unfavourable in membrane spanning proteins | Inserting polar groups into hydrophobic lipid bilayers is energetically unfavourable (enthalpic penalty) |
| α-helices non-covalent bonding pattern | Hydrogen bonding between residues i → i+4 |
| key structural features of transmembrane α-helices | ~3.6 residues per turn & ~20–25 residues span the membrane |
| Porins | β-barrel proteins allowing passive diffusion of small molecules, often in outer membranes & often with hydrophilic centres |
| Why β-barrels usually have even numbers of strands | Hydrogen bonding requires strand pairing, favouring even numbers |
| How hydrophobicity plots predict membrane proteins | Hydrophobic peaks (from averaging hydrophobicity of 7–11 residue windows along the sequence) indicate transmembrane domains |
| Membrane interface (“capping”) regions | Contains amphipathic residues for interacting with lipid headgroups & cholesterol |
| Step 1 in membrane protein study | Expression in the cell & trafficking to cell membrane |
| Step 2 in membrane protein study & typical protocol | Membrane isolation: cells are disrupted (e.g. sonication), debris removed, then membranes pelleted by ultracentrifugation (~100,000g) |
| Step 3 in membrane protein study & typical protocol | Solubilisation: Membrane proteins are hydrophobic & must be solubilised by detergents to remain in solution |
| Detergents | Amphipathic molecules with hydrophobic & hydrophilic regions |
| Critical Micelle Concentration (CMC) | The concentration at which detergents form micelles |
| Why micelles form above the CMC | Aggregation reduces free energy by shielding hydrophobic regions from water & makes the product more thermodynamically stable |
| How micelles solubilise membrane proteins | They surround hydrophobic regions & interact with water, replacing lipid interactions. They're often amenable to chromatographic separations |
| Detergent subtypes | Ionic: harsh, fully denaturing Non-ionic = mild, only denatures hydrophobic interactions zwitterionic = intermediate, denatures hydrophobic interactions & some protein-protein interactions |
| Detergent efficiency testing | Compare protein in supernatant vs pellet using western blot |
| Why some detergents fail | Hydrophobic mismatch or denaturation of protein structure. Specific proteins may require specific detergents |
| Step 4 in membrane protein study & typical protocol | Reconstitution: diluting or removing detergent below CMC to initiate a micelle to bilayer transformation & the membrane proteins will enter this |
| Reconstitution methods (2) | Dialysis: Detergent passes through dialysis tube into solution by osmosis but proteins stay behind Polystyrene: Detergents bind poorly to polystyrene beads but lipids bind well so you can remove detergents this way |
| Advantages & disadvantages of E. Coli expression system | High yield (~5% cell weight) but poor folding, proteolysis, secretion, & no post-translational modifications |
| Advantages & disadvantages of yeast expression system | Intermediate protein production & secretion with some PMT capabilities & sometimes 30% yield |
| Advantages & disadvantages of mammalian expression system (+insect cell expression) | Highest quality of yield but very low yield. Insect cells don't have the same production accuracy but 30% yield |
| Baculovirus | Enables high-yield protein expression in insect cells with some PTMs |
| Difficulties purifying membrane proteins | Micelles alter protein size, mask tags, & affect chromatography behaviour |
| Why membrane proteins must be reconstituted | Long-term detergent exposure destabilises proteins & disrupts function |
| SMA "cookie cutter" nanodiscs & disadvantages | Styrene-Malic Acid polymers that extract membrane proteins with surrounding annular lipids intact. But they're very sensitive to pH & divalent ions, which most biological buffers contain |
| How large amounts of specific membrane protein were hisorically gathered | Harvest from natural sources which were high in the protein of interest (e.g., VGCs from stingray electic organs). This was inefficient & often unethical |
| Why some reconstitutions can fail & how SMAs help | Some proteins require very specific lipid balance (e.g., mitochondrial proteins may need cardiolipin) to be native & SMAs can remove annular lipids for analysis |
Created by:
Denny12
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