Folding of Integral Membrane Proteins PC Flashcards
Name 2 diseases associated with membrane protein folding
Retinitis Pigmentosa-degenerative blindness caused by a misfolding in your vision receptor
CF-folding problem with Cl- channel/transporter
Problems with membrane folding?
Membrane proteins are hydrophobic
if you remove them from the bilayer they will non specifically aggregate
Difficult to isolate sufficient amounts in a stable state
Must study in complex membrane or micelle systems
Complicates almost all spectroscopic measurements using detergents and micelles as these scatter light like crazy
Very difficult to design and interpret folding expts in the absence of the knowing what the 3D structure looks like - having an electro crystal structure is useful but still not routine for membrane proteins
Can be challenging to identify unfolding conditions and denaturants
Does the protein fit anfinsen hypothesis?
Does the protein need translocon machinery for folding?
Reversibility of folding
What secondary structure are integral membrane proteins?
Membrane proteins are in about 2% of all protein structures in the world
Either all alpha or all beta
Aplha-receptors, transporters, channels, energy generating machinery
Beta-simple pores
When do detergents form micelles?
Above a specific concentration threshold : The critical micelle concentration (CMC)
Detergent micelles can be denaturing or renatruring (can fold and unfold protein-particularly with regards to an alpha helical membrane protein
Mixed micelle have been used for unfolding helical membrane proteins
What denaturants are used with alpha or beta proteins?
Alpha helical-often resistant to GdnHCl/Urea
Beta barrel-can be sensitive to GdnHcl/Urea
Cant use same denaturants people employ to study soluble proteins
Is the ribosome/translocon (SRP) machinery essential for folding membrane proteins?
Arguments are either way-are we being too reductionist
Are our simple in vitro methods too simple?
Will we ever truly understand this process without actually introducing a bit more complexity?
Do membrane proteins obey anfinsen hypothesis?
Or do they rely on other cellular machinery to help them along with their folding pathway
Does the general model of a ‘folding funnel’ apply?
Not sure whether they do sit at the bottom of an energy well or not
Does the protein have a lot of conformational freedom sampling lots of different states until it reaches its stable 3D structure
Reversibility of folding beta barrel proteins? Adding and removing GdnHCl
Without hysteresis as not good for studying protein folding:
Low pH
Slow Dilution
Small amount of detergent
Helps the protein refold along the path/trjectory upon it was unfolded (truly reversible process)
What is the three stage model for protein folding?
1) insertion
2) helix association
3) loop rearrangment/cofactor rearrangment
useful for studying protein folding-can study any one of those stages in isolation
Why are bacteriorhodopsin a good example organism?
What species of Archea is it for?
How many helices does it have?
What wavelength of light does it absorb?
Forms a trimer in the purple membrane and these trimers are naturally packed together
Membrane is bursting with bacteriorhodopsin
purple when folded optically clear when unfolded very stable and east to handle high res X ray structures available Can make targeted mutations
halobium salinarum -grows in 4M salt so no need for antibiotics as nothing can be contaminated with this high salt concentration
7 TM alpha helices
560nm - retinal chromophore with intrinsic spectroscopic signal
What detergents have been used with bR?
possible to fold and unfold bR in mixed micelles
Unfolding in bR mixed in DMPC (lipid) and CHAPS (cholesterol mimic) and SDS micelles
Upon addition and removal of SDS bR can unfold and refold. (reversible without hysteresis)
Modest unfolding-disruption of tertiary structure (helices spread apart) and ‘fraying’ of helix ends
TWO stage unfolding - no hysteresis
Confirmed with exponentials with unfolding a refolding expts
Unfolded does not mean a random coil peptide chain by any means
