TB3 - NMR

Question 1

How does NMR collect data?

Answer 1

Data collection relies on placing the sample inside a powerful magnet, sending radio frequency signals through the sample, and measuring the absorption of those signals. Depending on the environment of atoms within the protein, the nuclei of individual atoms will absorb different frequencies of radio signals. Furthermore, the absorption signals of different nuclei may be perturbed by adjacent nuclei. This information can be used to determine distances between nuclei and thus give the overall protein structure.

Question 2

What happens when a nucleus with a magnetic moment enters a strong magnetic field?

Answer 2

it will begin to precess like a spinning top, whereby the magnetic moment wants to align with the magnetic field. If the sample placed in this magnetic field is irradiated with radio waves at the same frequency, an NMR spectrum can be obtained.

Question 3

What is Larmor precession?

Answer 3

When a nucleus with a nuclear magnetic moment is placed in an external magnetic field, B0, the magnetic field of the nuclei will not simply be oriented opposite to the orientation of the magnetic field. Because the nucleus is rotating, the nuclear magnetic field will instead precess around the axis of the external field vector.

Question 4

Equation for ω

Answer 4

ω = γ B0

Question 5

Equation for ΔE

Answer 5

ΔE = hv = ℏ γ B0

Question 6

How do we observe the magnetic moment?

Answer 6

In order to observe the nuclear magnetization, we want to bring it perpendicular to the applied field. Applying a radio frequency pulse, which is perpendicular to the external magnetic field, can do this. If the pulse has the same frequency as the Larmor frequency of the nuclei, the magnetization can be directed from the direction of the magnetic field to a direction perpendicular to this.

Question 7

Explain bulk magnetisation.

Answer 7

In general, a typical NMR sample contains nearly Avogadro’s number of spins. In the presence of an external field, each of these dipoles will have random orientations along the x-and y-axes, but one of two orientations along the z-axis. The collective behavior of these spins is given by the vector sum of the individual magnetic vectors, known as the bulk magnetization, M0.

Question 8

What is the minimum sample required for NMR?

Answer 8

300μM

Question 9

Explain free induction decay

Answer 9

The nuclear magnetization perpendicular to the magnetic field will decrease with time, partly because the nuclear magnetization gets out of phase and because the magnetization returns to the direction of the external field. This can be measured as a frequency and a decay rate as a function of time, referred to as the FID.

Question 10

How is sensitivity increased in NMR?

Answer 10

using stronger magnets

Question 11

How do you go from the FID to an NMR spectrum?

Answer 11

FID is a function of time, so applying a fourier transform converts this to a function of frequency (NMR spectrum).

Question 12

What is chemical shift?

Answer 12

When a molecule is placed in an external magnetic field, this will induce the molecular electrons to produce local currents. These currents will produce an alternative field, which opposes the external magnetic field. The total effective magnetic field that acts on the nuclear magnetic moment will therefore be reduced depending on the strength of the locally induced magnetic field. This is known as chemical shift.

Question 13

How does lower shielding impact ppm?

Answer 13

It causes a higher ppm as it’s more exposed to the external magnetic field

Question 14

Why are additional pulses with differing times introduced in NMR?

Answer 14

These create extra dimensions (time dimensions) to the spectrum.

Question 15

Define J-coupling

Answer 15

Two nuclei in a molecule, which are connected by one, two, and three bonds, can be seen to be coupling in the NMR spectrum. The coupling is observed by a splitting of the NMR signal.

Question 16

What is the coupling constant?

Answer 16

The separation between the two components of the split J-coupled signal. Measured in Hz and helps define dihedral angles.

Question 17

What can coupling constants be used for in protein structure determination?

Answer 17

The three-bond coupling constant depends on the dihedral angle defined by rotation around the middle bond in the coupling system. The J-coupling may also be used to distinguish between trans and gauche conformations.

One particularly important application of the coupling constant is as a measure of the coupling between the Hα and the HN in the peptide backbone. This coupling depends on the φ-angle in the peptide bond. The coupling may also be measured by the coupling constant between the HN and Cβ.

Question 18

What are COSY spectra?

Answer 18

• ONLY SHOW THROUGH-BOND COUPLINGS
  The two-dimensional COSY spectrum is recorded so that the spectrum contains two types of signals: diagonal peaks and off-diagonal peaks, often called cross peaks. The diagonal peaks represent the signals from each of the 1H types in an amino acid. Cross peaks report the couplings between pairs of nuclei.

Question 19

What are TOCSY spectra?

Answer 19

By recording the correlation NMR spectrum in a way so that all the spins in a spin system of an amino acid are all correlated, you get a more complicated spectrum. However, this type of spectrum becomes very useful in particular in the heteronuclear NMR experiments.

Question 20

Why do we use 13C and 15N?

Answer 20

By introducing 13C and 15N, the spins in a protein are almost all being connected by one-bond couplings, facilitating the study tremendously.

Question 21

What is 1H-15N HSQC?

Answer 21

The 1H-15N coupling in the peptide bond is the starting point for the heteronuclear NMR analysis of proteins. This bond is present in every amino acid residue in a protein, except proline residues. The correlation spectroscopy method used to record this coupling is called a 1H-15N HSQC. Each ‘spot’ is an NMR signal representing the 1H-15N coupling from one of the residues in the protein, generating a ‘protein fingerprint’.

Question 22

What are sequential assignments?

Answer 22

This is a process by which a particular amino acid spin system identified in the spectrum is assigned to a particular residue in the amino acid sequence.

Question 23

Why can’t dipole-dipole interactions always been seen?

Answer 23

In solution, where there’s molecular motion, these interactions are averaged out so aren’t always observed.

Question 24

How can we get dipole-dipole interactions back?

Answer 24

In very strong magnetic fields, it’s possible to orient homogenously charged particles. Protein molecules placed in such environments will no longer have free molecular motion and the dipole-dipole interactions will be partially re-established. The degree to which the residual dipolar coupling is re-established can be controlled by the concentration of colloidal solution.

Question 25

What is cross relaxation?

Answer 25

Cross relaxation is a result of dipole-dipole interaction between proximate nuclear spins. When two spins are very close in space, they experience each other’s magnetic dipole moment. It’s possible by NMR pulse techniques to either reverse the direction of the magnetic dipole of the nuclear spin or to ‘turn off the magnetic dipole’ of one of the spins to measure how this affects the other spin. The rate by which this effect is transmitted to the other nuclear spin is the cross-relaxation rate, and this is inversely proportional to the sixth power of the distance between the two nuclear spins.

Question 26

Define the nuclear Overhauser effect

Answer 26

The nuclear Overhauser effect (NOE) is the transfer of nuclear spin polarization from one population of spin-active nuclei to another via cross-relaxation.

Question 27

What are NOESY spectra?

Answer 27

Used to determine the NOE, measured by the intensity of the cross peaks.

• ONLY MEASURES <5 angstroms

Question 28

What creates the size limitation of NMR?

Answer 28

The slow tumbling of larger molecules causes the NMR signal to broaden and decay quickly (it has a very fast relaxation time). When the relaxation time of the NMR signal occurs faster than the delays needed to run the experiment, the experiment will fail .

Question 29

Define correlation time, τC

Answer 29

Characterizes molecular tumbling in solution: the larger the molecule the slower it tumbles. Relaxation time is dependent on the correlation time of the molecule.

Question 30

What is stimulated annealing?

Answer 30

The repeated heating and cooling process, known as stimulated annealing, is meant to help energetically unfavorable structures to overcome energy barriers and end up in more energetically favorable structures, which may resist the subsequent heating process.

Question 31

Define RMSD

Answer 31

Resolution in NMR is based on the overlay of structures that are consistent with experimental restraints. This gives an RMSD, where the higher it is, the more structural variability.

Question 32

Chemical exchange: the process of slow exchange

Answer 32

In a sample where a ligand is in slow exchange with the protein and the signal of a given nucleus has a chemical shift for the bound and free conformations, NMR will record the sample as containing two different species: the bound and the free form.

Question 33

Chemical exchange: why does application of heat broaden signals?

Answer 33

This is because the nucleus is rapidly transferred from one magnetization condition to another, leading to line broadening. The line broadening effect is so strong that the NMR signal essentially disappears.

Question 34

Chemical exchange: the process at high exchange rate

Answer 34

At even higher exchange rates, the transfer of magnetization is faster than the difference in the chemical shift frequencies of the two different exchange sites. The magnetization will not precess with either frequency but will be observed as an average. The position of the average chemical shift depends on the fractions of the time the nuclear spin spends in each of the sites.

Question 35

Why is hydrogen often replaced for deuterium?

Answer 35

The rate of hydrogen exchange depends on the frequency by which the hydrogen bond is opening. By replacing hydrogen with deuterium, 1H NMR can study the exchange process since the hydrogen giving rise to a 1H NMR signal is exchanged by deuterium which isn’t observable by NMR.
By recording the hydrogen exchange rates, the stability of the individual hydrogen bonds can be measured. This can be done for every single peptide group of a protein and be used to describe the dynamic properties.