Nmr Flashcards
What is type of spectrum does the NMR experiment give
A 2D spectrum
What is the protein sample in an NMR experiment
What atoms are usually put in the sample and why
The sample has a magnetic moment or spin
Usually use the H isotope of your doing proton nmr
The sample is grown in media with enriched specific isotopes for labelling
These isotopes have a spin of 1/2 spin of one can’t be detected
How big is the sample for nmr
How much protein in the sample
0.5mL
1-2 mM (high concentration of protein)
Is nmr destructive or non destructive
What does this mean
Non
You can reuse the sample after analysis
What is the NMR magnet characteristics
500 to 800 MHZ
It’s a superconductor so it needs to operate at low temp
Has He (l) to cool the magnet at 4 K
And N2 (l) at 77 K (boiling point temp, keeps the He cold)
How does an NMR work
When there is no magnetic field, the HCN atoms randomly orient and spin
When there is a magnetic field applied, the atoms orient themselves in the direction of the field (parallel) or against it (anti)
When a pulse is applied, they become antiparallel to the field
When the pulse is gone, the atom precess back into their parallel state
We measure the spin of these to get the spectrum, the resonance frequency depends on the spin
In an nmr spectrum, if it shows H aplha what is it
The H on an alpha C
In H NMR what does the chemical shift of the leaks depend on
The environment of the hydrogen (whats orotund the H)
How many amino acids long is the c term domain of cellulase
39
In a chemical shift vs chemical shift diagram of the NMR spectrum what is observed (2D) spectrum
Dots on 2D spectrum diagonal axis show matches between the peaks of both the 1D spectrums
Each dot is a peak
What does a multidimensional NMR spectrum with H and N15 show us
The hydrogens that are attached to N atoms
What is COSY
Correlation spectroscopy
The Hydrogen is covelently connected through 1 or 2 atoms to another hydrogen atom
What can cosy help with
Since the H is connected through 1-2 atoms, you can see a fingerprint of the amino acid residues since the peptide bond separates one residue from the next
What is NOSEY
What does it helps with
It shows the H that are close together in space (not close together through bonds)
Helps determine secondary structure/folding of the protein
Helps identify adjacent residues
What are the steps in finding protein structur using NMR
Prepare the protein sample
Record the spectra
Assign peaks for back bone atoms
Assign peaks for side chains
Collect structural restraints
Calculate and refine the structure
Do follow up experiments
What are the experimental results of an NMR experiment
You can find the distances (distance restraints) between protons
from that find all possible secondary structures to make an ensemble of structures (superposing all possible structures)
How do you compare the structures that you got from NMR
RMSD
Less than one is good
If there are no hydrogen restraints on the amino tail what does this mean
Could be an intrinsically disordered region or we just don’t have the data for that region
Protiens for NMR analysis have to be ______
Why is this good
Soluble
Have to stay in solution for the entire experiment
Good because can suspend the protein in a solution that mimics its natural environment (like membrane proteins in a non polar solvent)
Also good for measuring dynamics because the proteins can move around in solution
Why would you do d2l exhange in NMR
Since deuterium has a spin of 1and Not half, it isn’t measured in the nmr
This helps because is sponges the signal that would have been given by water in the nmr experiment which reduced the noise
