Lec 31 Flashcards
What are the key methods to find folded and unfolded states of protiens
Hydrodynamic methods
Spectroscopic methods
HD exhange
Why do we used these methods to find folded vs unfolded
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
How Is a hydrodynamic method ( transverse urea gradient gel electrophoresis) used to evaluates protein folding
What is the weakness of this method
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
How can a hydrodynamic method ( transverse urea gradient gel electrophoresis) be used to probe binding of a ligand
How do you do it
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
How Is a hydrodynamic method ( transverse urea gradient gel electrophoresis) tell us the hydrodynamic radius of the protien
As the protein unfolds with higher urea concentration, the hydrodynamic radius is bigger and the protein is higher up in gel
What is molar ellipticity
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
Explain what CD spec is
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
Explain the Far UV secondary structure plot of a CD spec
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
Explain the far uv CD spectra of RNase A
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
Is the far IV spectra of RNase A why do we see the native peak going from -10,000 back to zero
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
Explain the near UV spectra of native and denatured RNase a
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
The more aromatics in near UV cd spec the ____
the more the signal goes down
What is intrinsic trp flourensce
What is the downside
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
What is the wavelength of emmision max for buried, surface, exposed, residues of trp
Buried: 320-330
Surface: 340
Fully Exposed (denatured): 350
Explain how intrinsic trp Fluor can probe drug binding
If drug is binding to the trp, it’s fluoresces shifts right and the halfway point is the apparent KD
In an intrinsic trp Fluor plot with increasing amounts of urea how can you see at what point the protien is getting more unfolded
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
What does the diff pH show us in intrinsic trp Fluor sense
Idk slide 9
Does the y axis in intrinsic Fluor matter
It’s emmision intensity,
No , just looking at if high or low
Explain the two state curve of molar ellipticity and intrinsic trp fluor
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
Is cd spec far uv what value do we see for mix , alpha helix beta sheet Randi coil
-10,000
-30,000
-20,000
0
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
The protein has only one transition/intermediate states when folding
Many transition states
What is ANS fluorescence
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
What is the complication of ANS fluorescence
How do you get over this
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
How does ANS fluor work
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
What can be used as denaturants
Heat, pH, chaotropic salt
Explain a three state folding curve with the far uv and near uv and ANS fluorescence
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
