Session 1.3: Membrane proteins Flashcards
What are the function of proteins in membranes and evidence for proteins in membranes?
Facilitated diffusion
Maintain ion gradients
Allow specificity of cell responses
What are the biochemical evidence for proteins in membranes?
Membrane fractionation and gel electrophoresis
Freeze fracture
How does electrophoresis work
SDS-PAGE - detergent which coats proteins with a negative charge
All move in same direction
Electrical gradient formed so negative proteins migrated into the gel
Smaller protein - travels further
Banding pattern forms
Stained to see bands and proteins identified, Eg: in red blood cells
How does freeze fracture work
A Crystal with cell within it
Press sharp knife against the crystal, progressively harder until it fractures (between 2 lamellae of the phospholipid bilayer)
So half membrane lipids to E fracture (in extracellular water)
Half to P fracture (in cytoskeleton)
Low angle shadowing, electron dense material fired and build up against proteins and will fill holes (works as density within a protein is high)
How do proteins in bilayers move
Conformational change - vibrational, to signal inside a cell that a signal has been received
Rotational
Lateral
No flip flop - amount of hydrophilic exposed, unable to flip
How are proteins restricted in their mobility
Aggregated/scaffolded - form interaction between proteins
Tethering - membrane proteins adhering to proteins in the substratum
Or interaction with cytoskeleton
Interaction with other cells
Proteins read to separate out into fluid phase or cholesterol poor regions
How do we describe membrane proteins
Peripheral - bound to surface, electrostatic and H bond, removed by changes in pH or ionic strength (salt concentration)
Integral - interact with hydrophobic domains of bilayer, cannot be removed unless by agents that compete for non polar interactions like detergents and organic solvents
What is the model of lipid membrane structure
Lipid mosaic model
Intergral membrane proteins through bilayer - Hydrophobic structures within them which stabilise their interaction with the hydrophobic domain of the membrane
Extra membranous regions such as extracytoplasmic surface and cytoplasmic surface
Peripheral proteins held by charged and h bonds
Can be completely integral
What are the amino acids present on the transmembrane part of the integral protein
Small - glycine
Hydrophobic - phenylalanine, leucine
Polar, uncharged - tyrosine
Often alpha helical (20-22 amino acids)
What is a hydropathy plot
To work out if a protein is a membrane protein - 20-22 amino acids and alpha helical
Apply 20 amino acids to an amino acid sequence - is the sequence hydrophobic or hydrophilic, window moved down from N terminal - if hydrophobic sequence around 20 amino acids = transmembrane, can be multiple transmembrane sequences
Glycophorin - single transmembrane domain orientation?
100% of the molecules are facing in the same direction
End terminal in cytoplasm and C terminal in extracellualr space
Carbohydrate always directed to outside
What is membrane protein topology
orientation of the protein in the bilayer
What are the different ways proteins can associated with bilayers
Single or multiple transmembrane (in and out of membrane) - integral Peripheral protein (one side or other) Postranslational lipid modifications - attaches to restrict movement or integral protein due to fatty acid modification
How are erythrocyte membrane maintained?
Cytoskeleton
How do you remove peripheral proteins to work out how much of the other membranes are left? (Intergral)
Add salt wash
Low angle shadowed with electron microscope what do you see in spectrin?
Alpha and beta chain coiled around each other
Rigid structure - part of cytoskeleton
What is the function or spectrin and where is it found
Proteins that hold the structure in place
At junctions, proteins that glue the lattice together and attaching it to membrane
Lattice attached to membrane
Identify - looks like a grid
What is the erythrocyte cytoskeleton composed of
Spectrin dimers joined end to end to form the lattice structure of the cytoskeleton
The lattice is held in place by gluing proteins - ankyrin (glues spectrin to band 3)
Band 4.1, actin and adducin (attachment proteins)- glue spectrin to glycophorin A (transmembrane)
What happens if there is an issue with proteins within the cytoskeleton of erythrocytes
Hereditary spherocytosis : - as spectrin depleted by 40-50% - erthryoctes round up instead of biconcave - less resistant to lysis so release Hb - cleared by spleen, depleted no of RBC in circulation, less O2 transported Hereditary elliptocytosis: - defect in spectrin molecule - unable to form heterotetramers - fragile elliptoid cells
How are secreted proteins synthesised
Ribosomes attach to 5’ prime end of RNA in cytoplasm and reading the triplet code, tRNA/AA complex released to add to protein -translation
Secreted proteins have a growing polypeptide at terminus - these are recognised by the signal recognition particle, which is recognised by a protein in the endoplasmic reticular, a docking protein - the whole complex of RNA and protein to the surface of the plasma reticulum
The growing protein made of lots of RNA is held physically against the ribosome, the ribosomes is thus unable to continue to elongate that protein
Leaves a free end terminal which can then be recognised by a single sequence receptor within the membrane
Starts passage through the membrane
Once removal of signal recognition site, particle synthesis can continue
How are membrane proteins synthesised
Signal expressed and synthesis begins
The signal is released from the signal recognition site and passed on to the signal sequence receptor
Synthesis continues until hydrophobic stop transfer sequence
If transmembrane - hydrophobic part of protein would rather form an interaction with the membrane - stop transfer sequence, locking the protein into the membrane
The ribosome detached and completes the rest of the protein in the cytoplasm so forms transmembrane protein with end terminal in the ER (positive charges attach to SSR, like a hairpin enters) and C terminal in cytoplasm
Peripheral proteins
Contribute to form the lattice structure of the cytoskeleton
How membrane proteins are inserted into membranes
Targeting of their synthesis to the membrane during synthesis
So inserted into the endoplasmic reticulum during synthesis - maintains orientation of membrane proteins
