Lecture 3- Membrane Proteins Flashcards
functional evidence for proteins in membrane
Channels and transporters - facilitated diffusion - ion gradients - specificity of cell responses
biochemical evidence for protein in membrane
- membrane fractionation and gel electrophoresis - freeze fracture
example of cell fractionation and feel electrophoresis of erythocytre membrane
(no organelles) 1. breaking up of cells e.g. with detergents 2. centrifuge 3. electrophoresis on SDS-page
electrophoresis separates proteins based on
size and charge - smallest migrate furthest towards positive electrode
freeze fracture is a technique
, a specimen is frozen rapidly and cracked on a plane through the tissue. This fracture occurs along weak portions of the tissue such as membranes or surfaces of organelles.
freeze fracture process
- Take cell and freeze- Locks cell membrane in position 2. Use sharp knife to fracture ice crystal 3. Fracture occurs where there is points of weakness- between bilayer 4. Can look at both sides of the phospholipid bilayer 5. Preparation can then be looked at under TEM
freezing cell
locks cell membrane in position
freeze fracture shows how
dense the membrane is with protein (60%)
mobility of proteins (3)
-Conformational change - Rotational- fluid lipid environment -Laterally- like phospholipids (nearly as fast)
why can’t proteins flip flop
due to thermodynamics- would break membrane
when protein density within membrane is high what forms
aggregates of proteins are called RAFTs
RAFTs can
restrict protein motion
proteins aggregates found
in cholesterol rich regions
tethering of cells to basement membrane/ ECM/ cytoskeleton etc can
stop proteins move in and out of cells
lipid associated effects e.g. cholesterol rich regions are more packed
restrict protein mobility
restriciton of membrane mobility (2)
- RAFTs - Tethering - lipid associated effects
type of membrane proteins
1) Peripheral 2) Integral
peripheral proteins
Bound to the surface of the cell membrane- not studded in membrane
peripheral proteins are bound to the membrane by
electrostatic and hydrogen bond ionteractions
how can peripheral proteins be removed
by changing pH or ion solution
integral proteins
- Interact extensively with hydrophobic domains of the lipid bilayer - Found within the membrane
how can integral proteins be removed
i. Cannot be removed by manipulation of pH and ionic solution ii. Are removed by detergents that compete for non-polar interactions
hydrophillic part of protein will always be
on the outside of the lipid membrane
hydrophobic part of the protein will always be found
in the inside of the bilayer
transmembrane polypeptide strucuture
often alpha helical - R groups of amino cid residues I transmembrane are largely hydrophobic
what can be used to can translate proteins sequence of polypeptide and score the amino acids for Hydrophilicity or hydrophobicity
Hypdropathy plots
glycoproteins membrane topology
• End terminal of protein will be in the cytoplasm • Oligosaccharide will be on the outside of the cell
N terminus on the
outside fo the cell e.g. where the ligand binds - oligosaccharide
C terminus
end terminal of protein will be in cytoplasm
normal erythrocyte cytoskeleton influences structure
makes it biconcave- large surface area and can squeeze through capillaries
cytokseleotn maintains
RBC structure- spectrin lattice
what can be used to visual proteins in the erythrocyte cytoskeleton
electrophoresis - Peripheral membranes washed off (salt wash) - Desired proteins (spectrin) can be visualised on electron microscope
most important protein involved in the erythrocyte cytoskeleton
Spectrin - maintains RBC integrity
how is the spectrum lattice adhered to the cell membrane
- runs parallel to the cell membrane - Ankyrin binds spectrin to Band 3 (integral protein in cell membrane)
if something goes wrong with the formation of the spectrum lattice
- weakness in cytoskeleton will occur- reusing structural integrity o the Roc –> these RBC will be damaged in blood vessels and destroyed by the body leading to anaemia
Weakness in spectrin lattice leafs to two types of
haemolytic anaemia
two types of haemolytic anaemia
- hereditary Spherocytosis - hereditary Elliptocytosis
hereditary Spherocytosis
- Spectrin depleted by 40-50% - Erythrocytes round up- blow up - Less resistant to lysis - Cleared by spleen
hereditary Elliptocytosis
- Defect in spectrin molecule - Unable to form heterotetramers - Fragile elliptoid cells- rugby ball shape
secreted protein synthesis e.g. insulin
1) mRNA produced in the nucleus moves to the cytoplasm where it forms a complex with the two subunits of a ribosome 2) The mRNA begins translating the mRNA 3) The first part of translated is the signal sequence 4) The signal sequence recruits signal recognition particle (SRP), which halts translation. 5) The SRP- ribosome complex then docks at a receptor located on the ER (forming rough ER) 6) Translation is re-initiated and the polypeptide chain continues to grow via a transport channel into the lumen of the ER 7) Synthesised protein will then be transported via a vesicle to the Golgi complex for secretion or the lysosome 8) Signal sequence is cleaved and the SRP recycles once the polypeptide is completely synthesised within the ER
what about membrane transporters e.g. Na+/K+ transporters
Proteins targeted for membrane fixation (integral proteins) get embedded into the ER membrane and ER membrane fuses with the celllmembrane over time
