Honours lecture Flashcards
What are NMR and computer based molecular modelling limited to?
Relatively small molecules. X ray better for GPCRS cos theyre big
What’s the principle of X ray crystallography?
- Make a crystal of the molecule you are interested in
- Shine X rays through it and look at the diffraction pattern
- This will depend on the arrangement of atoms in the crystal so you should be able to translate it into a structural model of the molecule.
What are the problems with X ray crystallography?
- Need large quantities of proteins. Cells don’t usually make large quantities of proteins, so you probably need to use an expression system
- Proteins (unlike small molecules) don’t like to form crystals
- Membrane proteins don’t like to be in solution- proteins will clump together (forming hydrophobic bonds).
What are halobacterium halobium?
Archaea. Grow in very high salt concentrations.. eg The Dead Sea.
H. halobium generates energy from light sensitive pigment, bacteriorhodopsin. Forms very high density crystal like arrays in the membrane of H. halobium.
What;s the structure of baceriorhodopsin?
- crystallized and determined by X ray in 1990
- 7 TM domains
- Same pattern as GPCRs (but not a gpcr!)
What’s the homology modelling?
Align features of unknown protein with that of a known structure.
Try to fill in the non-aligned parts using computer modelling
What’s a superfamily?
Group of similar proteins that have evolved from a common ancestral protein
Give four of the gpcr families?
- rhodopsin
- glutamate
- secretin
- adhesion
How do protein families evolve?
Have one protein. duplicates. one part mutates, becomes a different subunit to the protein.
How do you aliign proteins?
Line up the primary sequences and try match the biggest number of amino acids. Sometimes there are gaps introduced because of possible insertions and deletions. try take those into account.
What is the closest relative to AChBP?
A subunit called alpha-7. But only 25% of their amino acids aree identical. However the amino acids involved in the ligand binding part of the protein are same in all the sequences. AChBP can then act as a template for alpha7
How was homology modelling of GPCRs done?
Initially used bacteriorhodopsin for the GPCR superfamily.
Now more mammalian GPCRs have had their structure solved, including 2 human neurotransmitter receptors. these are used as they’re more closely related to human proteins.
Which gpcr ones have been found? in order
- bacteriorhodopsin
- bovine rhodopsin solved 2000
- human ß2 adrenoceptor 2007
- human D3 dopamine receptor 2010
some others since
What are the GPCR classes/subfamilies?
Family A/1- rhodopsin like
Family B/2- secretin receptor
Family C/3- metabotropic glutamate
D-F/4-6- Others
What does family A- Rhodopsin include?
adrenoceptors
muscarinic AChR
rhodopsins
dopamine receptor
