Biological NMR

Question 1

What is the spin property?

Answer 1

This is the angular momentum where particles are represented as spinning around their own axis. They can be thought of as tiny magnets.

Question 2

What is the spin value?

Answer 2

Quantum mechanics tells us that spin has a value of 1/2

Question 3

When placed in a magnetic field what are the two possible energy states?

Answer 3

Alpha- a state that is aligned with the magnetic field and is the lower energy state.
Beta- the higher energy state where the spin is aligned against the magnetic field.

Question 4

What are the five parameters of NMR

Answer 4

1. Chemical shift
2. Relaxation times
3. Scalar coupling
4. Dipolar coupling
5. Nuclear Overhauser effect

Question 5

Define Chemical shift anisotropy

Answer 5

The electron cloud surrounding the nucleus has a magnetic field which can align or oppose the applied magnetic field. This can change the energy required for a spin state change. It is described as anisotropic because the chemical shift is constantly changing and if the molecules are moving fast enough an average of these chemical shift values will be detected.

Question 6

What is the difference between scalar coupling and dipolar coupling?

Answer 6

Scalar coupling- the interaction of spin systems through bond. This can lead to the splitting of peaks.
Dipolar coupling- this is a through space interaction of spin systems. It can provide information on structural features of the protein.

Question 7

Define T1 and T2 relaxation

Answer 7

T1 relaxation- This is spin-lattice relaxation whereby the bulk magnetization returns to the Z direction and the reestablishment of the equilibrium of the alpha and beta spin states.
T2 relaxation- transverse magnetization. This is the loss of spins in the xy plane.

Question 8

What are the four steps of 2D NMR?

Answer 8

1. The chosen nuclei is supplied with a pulse (90 degrees) to result in the bulk magnetization transfer to the xy plane.
2. Magnetization is given an evolution period to ensure magnetization can be transferred.
3. Further pulses can be supplied for mixing between different nuclei
4. A signal is recorded from the second time domain

Question 9

Give three reasons why line width can increase with molecular weight

Answer 9

1. overlap problems- as molecules increase in size the spectra becomes more complicated
2. Intensity decreases-the molecule will relax much quicker so the less data is recorded
3. Inefficient magnetisation transfer

Question 10

What is the strategy for the NMR backbone assignment using triple resonance experiments?

Answer 10

1. The peaks are assigned as each of the types of atoms they are. This is done by using the overlapping triple resonance experiments.
2. The side chains can then be assigned in a similar manner
3. Following this, structural information can be found such as NOE data, torsional angles and distances
4. Structures can then be predicted by using allowed angle stereochemical data to define secondary structure features of the protein of interest. Such tools include Karplus curve which looks at torsional angles and relates them to particular angles of secondary structure. TALOS can also be used to predict phi and psi angles from the chemical shift data. This indicates the secondary structure residues may be held in.