Membrane proteins 1+2 Flashcards
(43 cards)
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.
