Membrane proteins 1+2 Flashcards
What percentage of the 100,000 proteins in the proteome are membrane-associated?
20% (20,000)
What is a transporter?
A protein that regulates the flux/flow/import/export/transport of chemicals from one side of a biological memrbane to the other
what is the main difference between transporters and channels?
Transporters undergo conformational change and are a million times slower
How can the membrane act as a marker?
Infected cells digest foreign protein in proteasome. Resulting peptides transported to ER. Peptides loaded onto MHC (major histocompatibility complex). MHC marks the surface of the infected cell. Cytotoxic T lymphocytes recognise the flag and engulf the cell.
How are peripheral proteins attached to the membrane?
Electrostatic interactions
What is their make-up?
Hydrophobic core, hydrophilic surface
How are they removed and are they soluble in aqueous solutions?
High salt buffers, yes they are soluble
Why are membrane protein surfaces hydrophobic instead of hydrophilic?
They need to interact with the hydrophobic interior of the lipid bilayer, allowing them to span the membrane
What about the core of the membrane protein?
It is also hydrophobic
What is the interior of ion channels?
Hydrophilic
Why are hydrogen bonds needed at the membrane and what structures form?
They prevent exposure of polar backbone groups / hydrophilic atoms because polarity is unfavourable in the membrane environment. Secondary structures form.
What form do membrane proteins take up then?
Folded state across the membrane as alpha helices or beta sheets because they have lots of hydrogen bonds for stability
Where do hydrogen bonds form in alpha helices?
residue i, and residue i+4
Where are sidechains positioned on every helix?
Outside of each helix
Where are sidechains positioned in beta sheets?
Alternatively towards inside and outside of barrel
Why do bacteria predominantly have an even number of beta strands in their barrels?
Because the first and last strand will eventually align next to each other once they have gone up and down the barrel. Then, they can form a covalent bond with each other through NH3 and COO- groups. (more detail on paper)
What is the purpose of hydrophobicity profiles along the protein’s primary sequence?
Can show regions which are likely to form helices. Stretches of greater hydrophobicity can mean there can be more helices present
What is the first step in studying membrane protein structure and function?
Expression of protein itself. E.g - getting acetylcholine receptor from brain
What is an alternative method to this?
Heterologous protein expression (protein expression in a different organism where it is not made - e.g - yeast/mammalian cells etc)
Why are bacteria useful at heterologous expression?
Low cost, simple culture conditions
Why are bacteria not useful at heterologous expression?
Low yield due to lacking of post-translational modifications like glycosylation. Eukaryotic proteins may not express well. They have a codon bias. They have improper folding and make the protein insoluble.
What type of eukaryotic cell is preferred for heterologous expression?
Insect cell
What is this process?
Virus which is lethal to insects releases large quantities of protein to kill it. Replace the protein encoding gene with our protein of interest gene, insert into insect cells and extract the protein being produced.
What is the first step in preparing proteins from a cell membrane?
- Cell disruption - high energy sound waves / immense pressure from nitrogen gas to infate the cell and expand it. These methods can destroy cell components like the cytoplasm.
What is the second step?
- Purification - extract the protein from the lipid bilayer using detergents because we need the protein to solubilised
Why is SDS is used as a detergent ingredient, what is a pro and con of it?
It removes lipids but unfolds the protein
What is a common characteristic of different detergents?
Long hydrophobic regions and a small amount of hydrophilicity
What is the chemical difference between lipids and detergents?
Detergents only have one hydrophobic tail whereas lipids have two
What is the significance of this?
It allows detergents to be soluble in water
How do detergents work?
They disrupt the lipid bilayer by inserting its hydrophobic tail. They protect the target protein from aqueous solution by wrapping its hydrophobic tail around it.
What is meant by the Critical Micelle Concentration (CMC)?
The point at which the target detergent has broken up the membrane and solubilised the protein within
What does the detergent break the membrane into?
Micelles
Why is the protein now water soluble?
Because it is surrounded by hydrophilic head molecules
Monitoring protein solubilisation by centrifugation and gel analysis: What is the first step after the detergent is above the CMC with the protein membrane?
Centrifuge at 100,000g for 60 minutes
What does this determine?
Whether a pellet forms or not or just a supernatant. Pellet represents unsolubilised material and the supernatant represents detergent solubilised protein in micelles.
Then run a protein gel to determine which detergent to use
How can solubilised membrane proteins be purified?
Techniques like chromatography for separation
Why can’t ion exchange chromatography be used on charged detergents?
Because it is based on charges
What is an essential part of this process for the detergent?
It is above its CMC
What is reconstitution?
Putting the protein back into the lipid bilayer by separating it from the detergent
How are detergents removed?
Dilute with water
What is an example of a method used for reconstitution?
Dialysis and dilution - external buffer to the detergent and protein which is in a bag. Small pores in the bag allow detergent to exit and the protein is too big so it remains in the bag. Lipid is added to reform into the lipid bilayer.
Is it important which lipid is reconstituted with the protein?
Yes, annular rings of lipids act as ‘coats’ for the protein
