Membrane Proteins Flashcards
What is the functional evidence for proteins in membranes?
Facilitated diffusion
Ion gradients
Specificity of cell responses
What is the biochemical evidence for proteins in membranes?
Membrane fractionation + gel electrophoresis
Freeze fracture
Outline the steps of SDS PAGE of an erythrocyte membrane
Centrifuge - separates into red liquid and white membrane clumps
Denature membrane with SDS detergent
Give all protein a -ve charge
Electrophoresis gel filters protein according to size - smaller move further
Describe freeze fracture
Frozen
Sharp knife on crystal
Fractured between lamellae of bilayer
Some proteins come off on E fracture face
Some left on P fracture face (p fracture face still has cytosol)
Holes left where proteins was
What are the 3 modes of protein motion?
Conformational change
Rotational
Lateral
NO FLIP FLOP
What restricts membrane protein mobility?
- Aggregates - “raft” of proteins stuck together - restrict mobility (membrane protein associations)
- Might be tethered to membrane or cytoskeleton (associated with extra-membranous proteins)
- Might be attached to other cells
- Lipid mediated effects - proteins tend to separate out into the fluid phase or cholesterol poor region
Describe peripheral membrane proteins
Bound to surface
Electrostatic and hydrogen bond interaction
Can peripheral membrane proteins be removed?
Yes, by changes in pH (eg by doing salt) or changes in ionic strength
Describe integral membrane proteins
Interact extensively with hydrophobic domains of the lipid bilayer
Can integral membrane proteins be removed?
Cannot be removed by manipulation of pH and ionic strength
– Are only removed by agents that compete for non-polar interactions
e.g. detergents and organic solvents
Describe the amino acids found in the transmembrane domain
R groups of amino acid residues in transmembrane domains are largely hydrophobic
Small, hydrophobic, polar, uncharged
Often alpha helix in hydrophobic domain
Describe hydropathy plots
Used to examine the “hydrophobic/philic-ness” along the length of a protein
Hydrophobic regions = transmembrane domain - can predict where they are
WHat are come ways prtoeins can be attached to the membrane?
Post translational lipid modifications - lipid attachments can interact with the membrane
Transmembrane domain
Describe the association of spectrum with the membrane
Spectrin dimers form lattice through membrane - interact through bilayer and band 3
2rod like structures would around each other
The lattice of spectrum under the membrane helps keep transmembrane proteins in place
What holds spectrin in place?
Ankyrin between band 3 and spectrin, and band 4.1
Describe 2 haemolytic anaemia
Rare
Treated by giving blood
E.g. Hereditary Spherocytosis – Spectrin depleted by 40-50% – Erythrocytes round up - not biconcave – Less resistant to lysis – Cleared by spleen
• Hereditary Elliptocytosis – Defect in spectrin molecule – Unable to form heterotetramers – Fragile elliptoid cells - Miss formed spectrin
Describe secreted protein biosynthesis
How to get hydrophilic part of integral protein through membrane
Or e.g. To package insulin into a vesicle
Secreted proteins have a hydrophobic sequence of 10-20 AAs at N terminal - this is what is made first in the ribosome
This hydrophobic end sequence is called a signal sequence
Signal recognised by RNA protein complex = SRP, signal recognition particle
SRP sees signal. Binds to signal and also binds to ribosome
Locks new part of protein to ribosome so ribosome cannot continue synthesis
Synthesis stopped in cytoplasm
All comes down to ER where there is a docking protein
SRP bring ribosome synthesising protein done to docking protein by ER
Signal is then released from SRP and is bound by signal sequence receptor in bilayer
Signal feeds through protein translocation complex into lumen of ER
Now synthesis continues until insulin fed through
When synthesis finished, whole protein in lumen of ER
As signal starts to emerge into ER lumen, signal peptidase chops of signal hydrophobic sequence as it is no longer needed
Post trans mod - cleaved into a and b chains
How is a transmembrane domain formed?
Put through, sequenced until a hydrophobic sequence is reached
When that seq made, prefers to stay in memb than go in lumen
=STOP TRANSFER SEQUENCE
Ribosome continues to make protein, lifts off and continues to make protein in cytoplasm
Trans membrane domain
N term in lumen
C term in cytoplasm
What would happen in the presence of an N-terminal signal sequence in the absence of a signal peptidase cleavage site?
C terminal in ER lumen:
No cleavage
Ribosome keeps making protein as a growing loop - N terminal not pushed through
Result is C terminal in ER lumen
What happens if there is a start transfer sequence (internal signal) in the primary sequence with its +ve charges at the N term end?
C terminal in ER lumen - not cleaved, loop builds up
What happens if there is a start transfer sequence (internal signal) in the primary sequence with its +ve charges at the C term end?
No signal peptidase
Ribosome continues synthesis in cytoplasm
N terminal in ER lumen
Describe how multiple transmembrane domains could be achieved
Trans memb protein in either way
What happens with multi trans memb?
1) signal = first trans memb domain, then loop
2) second trans memb domain could be a stop transfer
3) third could be another signal or make 2 and then plug them in
Describe right side out and inside out vesicles
Leaky by changing osmotic strength Seal it again Membrane is coming from plasma memb into cell to form vesicles So what was outside becomes inside What was inside becomes outside In vesicle
