Lec 30 Flashcards
What is the NPC
Nuclear pore complex
Made of protiens called nucleoporins
Made up of 456 individual protiens and 34 nucleoporins
It’s the gateways that lets protiens and RNA in and out of the nucleus
How Is integrative structural biology done to find the NPC structure
Fit the coarse grains model into the low res cryo em map
Keeps fitting until gets highest score possible
Then get final structure of the open and closed state of the NPC
What causes the low resolution of the structures
The flexibility or complex/dynamic nature of the protien
What happens if proteins misfold
Many diseases
What triggers protiens to misfold
Mutations
Environmental factors combined with genetics of the person
Ex. If PKA pathway (triggered by stress) , stress can cause protein misfolding if genetics aren’t proper to handle stress
What is the first step of the Haber and anfinsen experiment
First we denature the ribonuclease A
with chaotropic salt urea (disrupts hydrophobic interactions to denature native structure)
and BME (reduces disulfide bonds)
What is the second step of the Haber and anfinsen experiment
Do two treatments
A: oxidize in native environments
first remove the urea and BME by SEC or dialysis (now in native environment)
Then expose the sample to oxygen to oxidize the cysteine residues to reform disulfide bonds
B: oxidize in denaturing environment
First remove BME (keep urea) Expose the sample to oxygen to oxidize
Then remove urea
What did the two treatments in the Haber and anfinsen experiment tell us
So what was the conclusion
Treatment a (native environment)showed that the ribonuclease a got restored to the native form and had normals levels of enzyme activity and spectroscopic signals
Treatment B (denaturing environment) showed that native ribonuclease a was not formed and had low levels of enzyme activity and non native spectroscopic signals
They said that the disulphides bonds were not forming in the correct way in the denaturing environment, causing misfolding
In a rainbow colour ribbon diagram structure what does blue and red mean
What is the structure of ribonuclease A
Blue is n term red is c term
Central beta sheet and 3 alpha helices
Why does treatment A make folding proper
If remove urea and BME first, the protiens is already moving into its native structure
Then when oxidizing it is more likely to form the correct cysteines because their already close to each other
Why does treatment B make folding improper
Since urea is still there it stabilizes the non native conformation of the protiens
Then when urea is removed and the protien is oxidized protien is trapped in the non native disulfide bond confirmation
What are the lessons for the haber and anfinsen experiment
The denatured state is very different from the native state
Denatured proteins can SPONTANEOUSLY fold (within milliseconds) to make the single native state depending on the solution conditions
Denatured proteins can misfold to make many non native states depending on the solution conditions
What is levinthals paradox
Give math example
MAIN POINT: There isn’t enough time for the protien to randomly go over each conformation to find the right one , But somehow the protien still folds properly in a short time
So the denatured form must actually form many intermediate states to get to the native state
Math:
The two phi and psi angles in each residue determine the folding path that the main chain takes
If a 100 residue protein was folding it can take : N^100 possible confirmations
N is the number of distinct phi and psi angles conformations possible and to the power of number of residues
This means we can have a HUGE number of conformations even at N=2
What pwrcatges or time does a denatured protein form the native and non native form
90% fold proper and native
10% fold incorrect non native
What does most folding pathways resemble and why
Funnels
Because many paths to get to the unique folded state
What the critical step in ribonuclease A folding
Removal of urea forming the intermediate structure
Why is the process of protien folding hard to examine
Hard to capture the intermediates of the protien during its folding
Explain the energy landscape of a proteins
The lowest point is the most stable form, so most negative delta G.
But there are hills (misfolded, higher energy)
And valleys (intermediate states) where the protiens fall in the non native form and have intermediate stability (generally less stable than native) and get stuck there
What happens when protiens in the energy landscape get stuck in a intermediate state
Usually they go down the valley to the native state without intermediates
But when intermediates are formed there are chaperones that help it get pushed down the hill
In terms of the folding of protiens what I special about each different protien
Some have bumpier runs where they get stuck in many different intermediates
Some have smoother runs where they go straight to native state
Each follows their own path
What do denaturatant like chaotropic salts and reducing agents help with in terms of folding experiments
Help us control the relative amounts of folded and unfolded states of a protien in solution
Why is it important to measure the amount folded vs unfolded states of a of the protien in solution
If we can find how it folds we can find how to treat the disease it’s misfolding causes
Can compare the misfolded form to the Polyphen score and see if it’s a disease causing
Do this by introducing a mutation into the protien that is accosiated with a disease, check if misfolded in comparison to native state
What is gunidinium
The functional group on arg
What are two examples of chaotropic agents
Urea and guanidinium
What do chaotropic salts actually do
They reduce the energetic penalty that results from exposed hydrophobic side chains to water because:
The salts eject out this water shell that protiens are normally surrounded by and replace the water with themselves
Interact with the water through h bonding and the non polar side chains through van der waals
Then slowly stabilize protien misfolding
