GroEL Flashcards
What are 3 uses of cryoEM?
- Getting 3D images of ‘native-state; protein structures in high resolution
- Getting 3D images of unique cellular structures by electron tomography (ET)
- Correlating electron and light microscopy (CLEM) to get the bigger picture
What are 3 techniques you can use to obtain the structure of a protein?
- X-ray crystallography
- NMR
- Cryo-EM
What are the advantages and disadvantages of X ray crystallography?
Advantages:
• gives atomic resolutions
Disadvantages:
• needs specific conditions to obtain crystals
• only one conformation is shown
What are the advantages and disadvantages of NMR?
Advantages:
• gives near atomic resolutions
• you can see dynamic processes
Disadvantages:
• proteins must not be too large (around 30kDa)
What is the resolution of Cryo-EM?
3-30 Angstroms
Why is ice made from fast-freezing needed in Cryo EM?
Ice is needed in electron microscopy as it fixes the protein into one conformation
Fast freezing is required to create vitreous ice – this is better for the sample and image – glass like so it doesn’t obscure the image and disrupt the structure
What type of image is formed in Cryo EM and why?
A phase contrast image.
This is because electrons are being diffracted (relative phase changes when it passes through sample)
What is the structure of GroEL?
symmetrical, double ring structure
7x60kDa subunits in each ring - each with ATPase activity
Apical, intermediate and equatorial domains
Equatorial has nucleotide binding site
Apical is where GroES and unfolded proteins bind.
How has CryoEM shown the structure of GroEL?
Chen et al 1994 = first visualisation of unfolded protein substrate (malate dehydrogenase) bound to mobile outer domains at one end of (chemically modified mutant) GroEL. Showed that nascent or stress-denatured protein chains bind to the apical segment, which has hydrophobic binding sites
How has CryoEM shown the function of GroEL?
Roseman 1996 - got structure GroEL alone and with GroES - with ATP, ADP and AMP-PNP. Showed ATP causes rotation. Low resolution due to ATP forming various structures. Used to overlay crystal structures which were not in physiological conformation.
What happens upon ATP binding in GroEL?
- Salt bridge stabilising the two rings together is broken
- T state to R state via an anticlockwise twist
- hydrophobic protein-binding sites buried
- reduced affinity for substrate
- weakens the ring-ring interface so they each behave assymetrically.
What did we learn from CryoEM maps of GroEL?
Substrates rotate to weaken substrate affinity, ATP binding can be communicated between subunits, ATP binding and hydrolysis allows asymmetry between rings which opens the cavity for next substrate
Who used the slow hydrolysing ATP?
Ranson et al (2006) used a mutant that hydrolyses ATP much more slowly.
What were Ranson’s conclusions about GroEL?
Trans ring on ADP complex accepts ATP and new substrate.
It then affects the cis ring by promoting the ejection of GroES and ligands from the chamber.
This ensures that GroES is bound during the folding step as its release is triggered by ATP binding to the opposite ring.
