Lec 30 Flashcards

1
Q

What is the NPC

A

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

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2
Q

How Is integrative structural biology done to find the NPC structure

A

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

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3
Q

What causes the low resolution of the structures

A

The flexibility or complex/dynamic nature of the protien

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4
Q

What happens if proteins misfold

A

Many diseases

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5
Q

What triggers protiens to misfold

A

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

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6
Q

What is the first step of the Haber and anfinsen experiment

A

First we denature the ribonuclease A

with chaotropic salt urea (disrupts hydrophobic interactions to denature native structure)

and BME (reduces disulfide bonds)

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7
Q

What is the second step of the Haber and anfinsen experiment

A

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

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8
Q

What did the two treatments in the Haber and anfinsen experiment tell us

So what was the conclusion

A

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

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9
Q

In a rainbow colour ribbon diagram structure what does blue and red mean

What is the structure of ribonuclease A

A

Blue is n term red is c term

Central beta sheet and 3 alpha helices

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10
Q

Why does treatment A make folding proper

A

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

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11
Q

Why does treatment B make folding improper

A

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

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12
Q

What are the lessons for the haber and anfinsen experiment

A

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

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13
Q

What is levinthals paradox

Give math example

A

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

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14
Q

What pwrcatges or time does a denatured protein form the native and non native form

A

90% fold proper and native

10% fold incorrect non native

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15
Q

What does most folding pathways resemble and why

A

Funnels

Because many paths to get to the unique folded state

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16
Q

What the critical step in ribonuclease A folding

A

Removal of urea forming the intermediate structure

17
Q

Why is the process of protien folding hard to examine

A

Hard to capture the intermediates of the protien during its folding

18
Q

Explain the energy landscape of a proteins

A

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

19
Q

What happens when protiens in the energy landscape get stuck in a intermediate state

A

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

20
Q

In terms of the folding of protiens what I special about each different protien

A

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

21
Q

What do denaturatant like chaotropic salts and reducing agents help with in terms of folding experiments

A

Help us control the relative amounts of folded and unfolded states of a protien in solution

22
Q

Why is it important to measure the amount folded vs unfolded states of a of the protien in solution

A

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

23
Q

What is gunidinium

A

The functional group on arg

24
Q

What are two examples of chaotropic agents

A

Urea and guanidinium

25
Q

What do chaotropic salts actually do

A

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