Session 2.2a - Lecture 2 - Membranes: Biological Function Flashcards
Slides 1 - 16
How are membrane proteins involved in the bilayer?
Biological function
Title
How do we get proteins into the bilayer?
Title
Describe the dynamics of a membrane bilayer.
It is not a polythene bag around the outside of the cell but a kind of dynamic environment of lipids moving around each other
What do I need to know about membrane proteins?
- What is the evidence for membrane proteins?
- How may membrane proteins move?
- Can membrane protein movement be restricted?
- How do membrane proteins associate with the lipid bilayer?
- How may membrane proteins contribute to the cytoskeleton?
- How are membrane proteins inserted into membranes?
- How is correct orientation of membrane proteins maintained?
What is the evidence for membrane proteins?
ILO It might be helpful just to give evidence for the fact there are membrane proteins.
• Functional
– Facilitated diffusion
– Ion gradients
– Specificity of cell responses
• Biochemical
– Membrane fractionation + gel electrophoresis
– Freeze fracture
How may membrane proteins move?
ILO We’ve discussed how membrane lipids can move, so let’s also discuss whether proteins can move in the membrane.
- Conformational
- Rotational
- Lateral
NOT FLIP-FLOP
Can membrane protein movement be restricted?
ILO And if they can move, can their movement be restricted, bc that might be important, as I said in the last lecture in our cell on the BM, we have diff regions of membrane, would be good if we put the right proteins in the right place for the right function.
Restraints on mobility:
• lipid mediated effects
proteins tend to separate out into the fluid
phase or cholesterol poor regions
• membrane protein associations (aggregates; tethering; neighbouring cell interactions)
• association with extra-membranous proteins
(peripheral proteins), e.g. cytoskeleton
How do membrane proteins associate with the lipid bilayer?
ILO So how do we membrane proteins associate with the lipid bilayer – do they stick on the outside, do they form a sandwich?
- Peripheral
- Integral
How may membrane proteins contribute to the cytoskeleton?
ILO Having done that, we’ll be able to talk about the membrane cytoskeleton – that is the structure, the protein structure that maintains the basic shape of the cell.
- Spectrin lattices
How are membrane proteins inserted into membranes?
ILO
- SS/SRP/DP mechanism
How is correct orientation of membrane proteins maintained?
ILO Orientation of membranes maintained – need receptor for insulin facing out, no good facing into the cell etc.
- Signal sequences
What’s the evidence for proteins in membranes?
Functional
– Facilitated diffusion
– Ion gradients
– Specificity of cell responses
• Biochemical
– Membrane fractionation + gel electrophoresis
– Freeze fracture
How do we know there are proteins in membranes just by thinking?
Bc there are specific functions in membranes, and we know specific functions are determined by proteins.
How does facilitated diffusion dictate evidence for proteins in membranes?
They are relatively permeable to some stuff, but they facilitate diffusion of other stuff, e.g. glucose uptake, allowing ions to move across
Give two examples of things membranes can FACILITATE diffusion of.
- Glucose (uptake)
- Ions
How do ion gradients dictate evidence for proteins in membranes?
There’s much more Na outside than inside, so there must be proteins involved in maintaining that gradient
Where is Na greatest - inside or outside the cell?
Outside
How does specificity of cell responses dictate evidence for proteins in membranes?
E.g. some respond to insulin, other ones won’t, so there must be some receptor proteins in some cells and not others, hence functional evidence..
What is the functional evidence for proteins in membranes?
- Facilitated diffusion (e.g. glucose, ions)
- Ion gradients (Na is greater outside the cell)
- Specificity of cell responses (e.g. some cells respond to insulin)
What is the biochemical evidence for proteins in membranes?
– Membrane fractionation + gel electrophoresis (of RBCs)
– Freeze fracture
Why do we use RBCs as evidence for proteins in membranes?
It is a simple membrane system as it has a plasma membrane but no organelles in the mature RBC.
How can we burst RBCs?
Dropping them into hypotonic solution.
What is a hypotonic solution?
When there is less SOLUTE in the solution (therefore, a cell in HYPOtonic solution will BURST because there is MORE solute in the cell, thus water will move into the cell).
Why do we put RBCs into hypotonic solution when performing SDS-PAGE of the erythrocyte membrane?
So it bursts, releasing its Hb and other cellular contents from its cytoplasm, ready for centrifugation.
How do we separate the erythrocyte membrane from the cellular constituents?
Spinning the mixture hard in centrifugation.
What does centrifugation of a lysed RBC leave you with?
- Red supernatant with all the Hb in it
- White pellet of membrane at bottom of the tube
What is the supernatant?
The liquid found in a tube that lies above a solid precipitate.
Why do we centrifuge before SDS-PAGE of the erythrocyte membrane?
To separate the erythocyte membrane from the cellular constituents.
What is the function of SDS?
- Detergent
- Denatures membrane
- Coats all proteins with negative charges
What do we use to denature the erythocyte membrane in SDS-PAGE?
Detergent called SDS.
What do we need to do to a protein for it to work in electrophoresis?
Coat it with a negative charge - SDS.
How is electrophoresis run?
Proteins are run through a gelatinous medium, with an electric potential across the gel. Proteins are covered in a negative charge and therefore will run according to size.
How are the proteins separated in electrophoresis?
The gel will filter effectively those proteins run according to size
- Small ones will run all the way through the gel
- Big ones will get stuck near the top
Describe the procedure after isolation of a RBC to how it can be separated to show SDS-PAGE of the erythrocyte membrane.
1) RBC is chosen because it has a SIMPLE MEMBRANE SYSTEM: it has an outer plasma membrane but no organelles (therefore no membranes) in mature RBC
2) RBC is dropped into HYPOTONIC solution so it BURSTS to release Hb and cytoplasm contents
3) This mixture is CENTRIFUGED, leaving us with a RED SUPERNATANT with all the HB in it (CELLULAR CONSTITUENTS - OP) and a WHITE PELLET of MEMBRANE (ERYTHROCYTE MEMBRANE - BOTTOM)
4) Membrane is DENATURED with a DETERGENT called SDS
5) SDS also coats proteins with a NEGATIVE CHARGE
6) Proteins can then be run on an ELECTROPHORESIS GEL - a GELATINOUS MEDIUM that has an ELECTRIC POTENTIAL across it
7) The proteins will separate out according to size - the LARGEST proteins will STAY nearer the beginning whereas the SMALLEST proteins will RUN FURTHER
Fig. 4
Explain what this figure is showing.
Electrophoresis of erythrocyte membrane proteins - proving evidence that there is proteins in membranes.
Small things are at the bottom and large at the top (see direction of electrophoresis) e.g. Actin is small (bottom), Spectrin is large (top)
What is Band 3?
An erythrocyte membrane protein
What is Spectrin?
A very large erythocyte membrane protein
How does membrane fractionation and gel electrophoresis give biochemical evidence for proteins in membranes?
Membranes can be fractionated (separated) and then run on a gel electrophoresis, which coats proteins in negative charges and separates them out for size, which is visible on a stain. As staining is for proteins, if staining occurs, then there is evidence for proteins.
Fig. 4
Caption and label this image.
SDS-PAGE of the erythrocyte membrane
Direction of electrophoresis (down)
- Spectrin a
- Spectrin b
- Band 3
- Glycophorin
- Band 4.1
- Actin
Label 6 proteins found in erythrocyte membranes.
- Spectrin a
- Spectrin b
- Band 3
- Glycophorin
- Band 4.1
- Actin
Draw the SDS-PAGE of the erythrocyte membrane.
See Fig. 4
SDS-PAGE of the erythrocyte membrane
Direction of electrophoresis (down, or corresponds with proteins shown)
- Spectrin a
- Spectrin b
- Band 3
- Glycophorin
- Band 4.1
- Actin
(Must be in this order - spectrin must be at top bc it is a large protein (1 mark)
Large band for Band 3 bc it is prominent
Approx. size and spacing of proteins).
What do we need to do to the cell in freeze fracture?
Freeze it to make a crystal
How do we fracture the cell in freeze fracture?
With a very very sharp knife pushing gently onto the crystal - the crystal ultimatelt fractures due to the pressure of the knife and crystal.
Why does placing a knife onto the crystal break the cell in freeze fracture?
Due to the pressure
Where are you aiming for the knife to fracture in freeze fracture of membranes?
Between the lamellae of the membrane, splitting it.
What does the freeze fracture fragment into?
Two pieces
- P fracture face
- E fracture face
What do the P fracture face and E fracture face show?
A sphere of frozen phospholipid with some proteins left behind and some pulled off, which will be shown on the converse fracture face (i.e. a protein will be pulled off on the P fracture face leaving a hole there, but will be found in the E fracture face and vice versa).
Why is nothing moving in freeze fracture?
Remember this is frozen so nothing’s moving at the moment.
Describe the process of freeze fracture.
1) FREEZE your CELL to form a CRYSTAL
2) Bring a very very SHARP KNIFE to bear on that crystal, and PUSH GENTLY gently gently - the crystal will ultimately FRACTURE due to PRESSURE of KNIFE and CRYSTAL.
3) If lucky, FRACTURE will be BETWEEN LAMELLAE of MEMBRANE, SPLITTING IT.
4) This leaves two fracture faces - P FRACTURE FACE and E FRACTURE FACE.
5) Some proteins are LEFT in the P fracture face and others PULLED OFF, leaving a HOLE, where the protein is found in the E fracture face, and vice versa.
6) The whole system is FROZEN so NOTHING’S MOVING.
Fig. 5
Label this image depicting freeze fracture.
Direction of fracture
- Transmembrane protein
- Lipid bilayer
- Ice
Fracture with knife
P fracture face
E fracture face
What is the P and E fracture face?
Extra detail
(1) . E face = inside of the monolayer that is closer to extracellular space (outside of cell)
(2) . P face = inside of the monolayer that is closer to protoplasm (inside of cell)
Draw a diagram depicting freeze fracture.
See Fig. 5
Direction of fracture
- Transmembrane protein
- Lipid bilayer
- Ice
Fracture with knife
P fracture face
E fracture face
Fig. 6
Explain the diagram.
Effectively what we’ve done is our knife has fractured down the middle of the bilayer and pulled two halves apart so we either leave protein in place or we have a hole where there was a protein that’s been pulled with the other face of the membrane
How can we visualise the freeze fracture preparation?
Via electron microscopy
Give an example of an electron-dense ion we use in electron microscopy.
Osmium
Osmium is an ______-_____ ___ we use in electron microscopy.
Electron-dense ion
How are electrons used in EM?
Electron-dense ions, like osmium, can low-angle shadow a preparation - showing blobs where there are proteins and dips where there are holes (a bit like a molecular snow-drift blowing against a fence).
Explain the process of freeze fracture electron microscopy, after the two fracture faces have been separated.
1) ELECTRON-DENSE ION is used, e.g. OSMIUM
2) This low-angle shadows the preparation, like a molecular snow-drift blowing up against a fence
3) Where there are PROTEINS you will see BLOBS, and where there were HOLES from the freeze fracture you will see DIPS
Fig. 6
Label this image depicting freeze fracture.
Plane of fracture (middle) Lipid bilayer (orange and red) Frozen cytosol (pink) Frozen extracellular water (Blue) Band 3 molecule (mainly in cytosol) Glycophorin molecule (mainly in extracellular water)
P fracture face (protoplasm, inside)
cytosol
E fracture face (extracellular, outside)
extracellular water
Draw a detailed freeze fracture preparation.
See Fig. 6
Plane of fracture (middle) Lipid bilayer (orange and red) Frozen cytosol (pink) Frozen extracellular water (Blue) Band 3 molecule (mainly in cytosol) Glycophorin molecule (mainly in extracellular water)
P fracture face (protoplasm, inside)
cytosol
E fracture face (extracellular, outside)
extracellular water
1 mark - showing splitting down the lipid bilayer
1 mark - two halves, one facing the cytosol, one facing extracellularly
1 mark - protein in one half with corresponding hole in the other half, vice versa depending on proteins shown
Fig. 7
What is significant about this freeze fracture?
It is not a sea of smooth lipid with an occasional blob in it, but the proteins are packed into the membrane - they are really highly represented within the whole structure
Why is it important to remember that proteins are packed into the membrane, and not occasionally siphoned in?
So when we start talking about communication between proteins in the membrane – not to imagine protein wandering around looking for someone to interact with, the interacting partner is likely to be right next door, waiting to work with them
Fig. 7
Where is the interacting partner for proteins in the membrane likely to be?
This figure shows the membrane is packed with proteins, thus, the partner is likely to be right next door to it - proteins are not occasional items wandering around looking for their partners - their partners are probably waiting there for them to work with.
What is the visual evidence that shows membranes are PACKED with proteins?
Freeze fracture electron micrograph
What is the biochemical evidence for proteins in the membrane?
- Membrane fractionation and gel electrophoresis (separate the membrane and run it on a gel, proteins will move according to size, thus showing many different types of proteins)
- Freeze fracture electron microscopy (shows proteins and shows that they are abundant in the membrane)
Fig. 6+
Label and caption this image.
Freeze fracture electron micrograph of human erythrocytes
P face
E face
[1 um]
Draw a freeze fracture electron micrograph of a human erythrocyte.
See Fig. 6+
Freeze fracture electron micrograph of human erythrocytes
P face (protrudes out) E face (dips in) [1 um]
1 mark - scale bar
1 mark - many blobs showing abundance of proteins.
Where are proteins found in membranes?
Studded fairly DENSELY throughout the bilayer.
Can proteins move?
Yes, there are 3 modes of motion permitted.
What are the 3 modes of motion possible for protein mobility?
- Conformational change
- Rotational
- Lateral
NOT flip-flop
Why do we need our proteins to be able to move?
Just as our PLs can vibrate, our proteins can move – that’s good bc if we have a transporter that needs to grab something and put it into the cell we’ve got the possibility of moving to do that function
Explain conformational change in protein mobility in bilayers?
Proteins have quaternary structure, so we know that they have a 3D structure and that 3D structure is not fixed, it can undergo conformational change – it can go from diff states, but that protein will tend to flicker between certain stable states/conformations
Explain rotation in protein mobility in bilayers?
Proteins can rotate – some proteins are free in the bilayer, sitting like ships in sea of lipid, can move around in lipid in that way.
Explain lateral movement in protein mobility in bilayers?
Similarly, as you would imagine, they can move laterally, chug along through membrane and move somewhere else by changing places with lipid.
What movement CANNOT occur in protein mobility in bilayers?
NO FLIP-FLOP
(cannot move to other side of bilayer - think rugby players from back of lecture jumping to front of lecture theatre in PL, but cannot do that for proteins)
What movement is not permitted in proteins in bilayers, and why?
Flip-flop
Bc we have so much hydrophilic structure in that part of the protein sticking out the outside of the cell (or into the inside of the cell - i.e. out the membrane) so the thermodynamic energy required to move it across the bilayer is too large, it just won’t happen, can’t put enough energy in, and even if it did happen think about this – if you start taking a big chunk of protein through the membrane you’re going to destroy the membrane and its integrity – no benefit to the cell to being able to chunk protein through a membrane via flip flop.
Give two reasons why a protein cannot flip-flop.
- Thermodynamic energy required to move proteins across bilayer is too large (large hydrophilic structure from proteins sticking out into cell/ECM needs to move across hydrophobic interior of the membrane)
- Even if enough energy was acquired, it will destroy the integrity of the membrane (protein is too large, thus no benefit to cell to flip-flop)
Proteins in the membrane are able to move but unable to flip-flop. What does this mean for the membrane?
So just as our PLs can vibrate, our proteins can move – that’s good bc if we have a transporter that needs to grab something and put it into the cell we’ve got the possibility of moving to do that function.
However, lack of flip flop means once protein is in membrane going to be fixed by orientation.
