Lec 31

Question 1

What are the key methods to find folded and unfolded states of protiens

Answer 1

Hydrodynamic methods

Spectroscopic methods

HD exhange

Question 2

Why do we used these methods to find folded vs unfolded

Answer 2

The structure when unfolded due to mutations gets destabilized (when destabilized it’s hard to see using cryo em for example)

Also the methods can show the binding of ligands to the proteins and give apparent affinity

Question 3

How Is a hydrodynamic method ( transverse urea gradient gel electrophoresis) used to evaluates protein folding

What is the weakness of this method

Answer 3

Have a gradient gel of chaotropic salt

Charge Migration vs concentration of chaotropic salt plot

So in native state (low urea) it is more compact migrates faster (lower in gel)

as more urea, misfolded, more extended shape , migrates slower (higher in gel)

Reports on global not local structure

Question 4

How can a hydrodynamic method ( transverse urea gradient gel electrophoresis) be used to probe binding of a ligand

How do you do it

Answer 4

Run different lanes in the gel and add increasing ligand concentration in each lane

Then you’ll see a shift in the transition state to the right because the structure get stabilized by the ligand binding

The transition state diffenrce in without ligand and with ligand give apparent kd

Apparent because not directly probing the binding sites, just protiens folding, indirect measurements

Question 5

How Is a hydrodynamic method ( transverse urea gradient gel electrophoresis) tell us the hydrodynamic radius of the protien

Answer 5

As the protein unfolds with higher urea concentration, the hydrodynamic radius is bigger and the protein is higher up in gel

Question 6

What is molar ellipticity

Answer 6

Molar shows how this method is concentration dependent

Ellipticity is How much of the polarized light is being rotated (right handed or left handed rotation) from the chiral molecules in the protien

MEASURING ROTATION OF LIGHT

Question 7

Explain what CD spec is

Answer 7

We have two modes

Far UV: usually done at 220 nm, but if want to do full scan you do it at 190-240 nm, tell secondary structure

Near UV: measures around 275 or 280 nm and tells about the amount of order in core aromatic residues like phe, tyr, trp

Question 8

Explain the Far UV secondary structure plot of a CD spec

Answer 8

Alpha helical: starts with Postive values on the plot at 190nm which means positive rotation (right handed). Then goes down to the very negative values (left handed rotation) and peaks at 220 nm

Beta sheet: not as high right handed rotation as alpha helix and goes down to less negative value (less left handed rotation). Peaks close to 220, lower rotation

Random coil (flexible loops): starts negative (left handed) then goes up close to 0 and peaks at 220 around zero

Question 9

Explain the far uv CD spectra of RNase A

Answer 9

The native form has a peak going from 0 to negative values peaking at 220 nm at -10,000, meaning beta sheet and alpha helix and random coil mixed

The denatured from has just from negative to the zero meaning random coil

Question 10

Is the far IV spectra of RNase A why do we see the native peak going from -10,000 back to zero

Answer 10

Since the native form is made up of all beta sheet random coil and alpha helix, the resulting peak is a sum of all these signals

Don’t get full negative 30,000 value because not purely alpha helical

Question 11

Explain the near UV spectra of native and denatured RNase a

Answer 11

The rotation of light is much more reduced since only looking at specific residues, so looking at smaller y axis range

The native form starts less negative then goes more negative, peaking at 275 nm at -200 then to zero (this means has phe tyr and trp side chains)

The denatured starts very negative then goes to zero, since denatured and now random conformation they don’t contribute to the rotation of light

Question 12

The more aromatics in near UV cd spec the ____

Answer 12

the more the signal goes down

Question 13

What is intrinsic trp flourensce

What is the downside

Answer 13

Looking at the fluorescence/emmision wavelength of trp to see its exposure to solvent

Since trp is usually buried in folded protiens, this is useful to see if the protiens denatured (so trp more exposed to solvent)

Very sensitive to the local environment around the trp

Question 14

What is the wavelength of emmision max for buried, surface, exposed, residues of trp

Answer 14

Buried: 320-330

Surface: 340

Fully Exposed (denatured): 350

Question 15

Explain how intrinsic trp Fluor can probe drug binding

Answer 15

If drug is binding to the trp, it’s fluoresces shifts right and the halfway point is the apparent KD

Question 16

In an intrinsic trp Fluor plot with increasing amounts of urea how can you see at what point the protien is getting more unfolded

Answer 16

So a zero urea, peak at 330 nm so native protien

As urea increases, peak shifts right. The peak that gives the most shift to the right (closer to 350nm) relative to the last one is where the protiens gets more unfolded. This is the concentration of urea that gives the most unfolding

At highest urea, peak at 350, fully unfolded

Question 17

What does the diff pH show us in intrinsic trp Fluor sense

Answer 17

Idk slide 9

Question 18

Does the y axis in intrinsic Fluor matter

Answer 18

It’s emmision intensity,

No , just looking at if high or low

Question 19

Explain the two state curve of molar ellipticity and intrinsic trp fluor

Answer 19

First, have increasing chaotropic salt (GdHCL) on x axis

on one y axis you have the Florence’s intensity at a wavelength of 350

With less salt, the intensity is low at 350 meaning native unfolded

With more salt, intensity is higher at 350 meaning unfolded denatured

Now with CD, if measuring at 220 nm it’s far uv, so looking at the secondary structures

At zero urea if -10,000 (-10 on plot) it’s a mixture of alpha helix and beta sheets and random coil

Then when denature it’s at zero at 220 nm, meaning random coil

Question 20

Is cd spec far uv what value do we see for mix , alpha helix beta sheet Randi coil

Answer 20

-10,000

-30,000

-20,000

0

Question 21

In a two state curve if the molar ellipticity and intrinsic fluorescence plots line up in a single s shape what does this mean

What happens if multiple inflection points

Answer 21

The protein has only one transition/intermediate states when folding

Many transition states

Question 22

What is ANS fluorescence

Answer 22

Measuring the fluoresce of ANS

In solution get low signal

If ANS in an exposed hydrophobic environment, get high flurorescent signal meaning more unfolded protien

Question 23

What is the complication of ANS fluorescence

How do you get over this

Answer 23

The binding of ANS can change the conformation of the protien because it stabilized a different conformation when bound to it

Usually combine this method with intrinsic trp fluorescence

Question 24

How does ANS fluor work

Answer 24

At low ph (less than 4) the protien is unfolded and has exposed hydrophobic patches

ANS bind to this and fluoresces a lot

when higher pH, protien folded, ANS can’t bind, no fluor

Question 25

What can be used as denaturants

Answer 25

Heat, pH, chaotropic salt

Question 26

Explain a three state folding curve with the far uv and near uv and ANS fluorescence

Answer 26

On y axis there is fluoresces on x there is salt

See increase in flour as salt goes up since unfolded and ANS is binding

Also the far and near uv shows two inflection points , meaning the protien has two different transition states/ folding intermediates