Lecture 4 - Membrane Proteins Flashcards
What is the functional evidence for proteins in membranes?
By observing cell function we know that:
Facilitated diffusion happens across membranes such as the movement of hydrophobic molecules must be assisted
Ion Gradients exist across membranes, so we must need an ion transport system
specific cell responses - will require protein receptors
What are the biochemical tests for evidence of proteins?
1) membrane fractuation and gel electrophoresis
2) Freeze Fracture
3) Hydropathy plots
Explain the process of gel electrophoresis
First the cell is spun in membrane fracturation, this leaves only the membrane as a ‘pill’.
the pill is treated with sds page to ensure all proteins are negatively charged.
the proteins are put on a porus gel between two electrodes , and they all move to positive electrode
the heavy molcules move slower, smaller faster. This is due to diffusion.
the process separates and indentifes proteins
Explain freeze fracture
the cell is frozen into a crystal
it is then shattered with a knife
the membrane will split into two
we treat (shadow on) an electron dense metal, that build up at proteins
we can now image the membrane with an electron microscope
How may membrane proteins move?
confromational changes
lateral diffusion in the membrane
rotation
they cannot FLIP FLOP, this requires too much energy
how does cholesterol restrict protein mobility?
proteins separate themselves into regions of more or less cholesterol
a region with lots of cholesterol is very rigid, a region with less is more fluid
most proteins tend to stay in the more fluid region of the membrane, however signalling proteins stay in the cholesterol dense regions
what are ways in which proteins are restricted within the membrane?
proteins can interaction and then aggregate together, this group will move less, restricting mobility
proteins can be tethered to proteins in/outside the membrane such as the cytoskeleton, which fixes them into place
if two cells are in a tissue they may have trans membrane channels to each other, so interactions with other cells can fix proteins in place
What are the ways in which membranes associate with the lipid bi layer?
there are peripheral proteins- these attach to the membrane with electrostatic interactions and H bonding
changes in the Temp, Ph, and ionic strength will remove these proteins
any proteins not removed by this are integral proteins, they interact with the hydrophobic domains if the bi layer
they are removed by agents that compete for no polar interactions such as detergents, but this also destroys the membrane.
they can also associate using dichol phosphate polypeptide
How do hydopathy plots work?
we know that the bi layer is roughly 50 A in length, we also know that to cross this takes appox 20 amino acids
we measure using hydropathy how hydro Phillic/Phobic the section of the protein is
it will always follow the pattern of phobic-phillic-phobic…ect
the number of hydrophobic regions that we see tells us if it is a single trans membrane domain or 7 transmambrane domains ect
what is the topology of proteins?
all proteins are always oriented one way in the membrane
what amino acids exist in the membrane bilayer?
the amnio acids that are within the hydrophobic section of bi layer are often small and hydrophobic themselves, they may be polar, but NEVER charged
what are postranslational lipid modifications?
they are additions to the protein after translation that may add for example a fatty acid, this can constrain the protein into a confirmation.
it does this by binding the fatty acid to the lipid membrane, in doing so it makes in a integral protein
what is a ghost membrane?
after centrifuge , all the cells contents has been spun out leaving only the membrane - appears white
what is the importance of spectrin within the erythrocyte cell?
what are the issues if it goes wrong?
spectrin proteins associate in an end to end lattice structure which is grafted to the membrane via attachment proteins - glycophorin and band 3 proteins through ankyrin
if it goes wrong you get heamolytic anaemias
hereditary spherocytosis and hereditary eliptocytosis
where the body produces less spectrin / spectrin is defected (sphere/rugby ball) this means the lattice is not formed properly, and so the erythrocytes wills undergo lysis in the capillaries and spleen, leading to anaemia
we treat this with blood tranfusions
how are secretory and membrane proteins synthesised?
a ribosome will begin to transcribe the protein
SRP will bind to a hydrophobic leader (signal) sequence, this stops synthesis.
the a docking protein will remove the SRP,
next to this is a signal sequence receptor (tranlocator complex) that will pull the protein into the endoplasmic recticulums lumen
it can now continue to be transcribed
it can now be released once complete and ribosome stops synthesising.
the process of scerection happens via the leader signal stopping in the membrane as its hydrophobic, then the protein folds into the mebrane, it is then cut via signal peptidase allowing the rest of the protein to pass through the membrane while the leader signal is left. the N terminus is released into the lumen
for membrane proteins the process is the same
but the hydrophobic section stops transfer when passing through the membrane, locking that part of the protein into the membrane
