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Imperial (Y2) - Structural Biology > Steven Matthews - NMR > Flashcards

Steven Matthews - NMR Flashcards

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1
Q

What are the main applications/uses of NMR?

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2
Q

What is Nuclear Spin?

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3
Q

For a spin ½ nucleus, what happens when we place the nuclear magnetic field into external magnetic field?

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4
Q

What does the energy level between these two states depend on? Can we manipulate the number of nuclei in these two states?

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5
Q

Is NMR as sensitive as other techniques that use higher magnetic fields ?

A

NMR is not as sensitive as other techniques that use higher magnetic fields

Because we are dealing with very small energy differences between spin states.

Example - The energy differences are 10-5 smaller than that associated with infrared.

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6
Q

Examples of frequently used spin nuclei, spin state and their relative sensitivity?

A

Proton has largest γ as it has the smallest nucleus

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7
Q

In the past how were NMR spectra recorded?

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8
Q

What does the Larmor precession of nuclear spins refer to?

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9
Q

How can we represent the net magnetisation of spin 1/2 nuclei in the presence of strong external magnetic field?

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10
Q

What happens to the net magnetization when we apply a radiofrequency pulse?

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11
Q

Describe what happens to the spin states in the images when a radiofrequency pulse is applied at 90o?

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12
Q

What is the relationship between the length of a frequency pulse and the number of frequencies it contains?

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13
Q

Outline the basic principle behind NMR?

Hint - Pulse, free induction decay and Fourier transform

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14
Q

What are the different parameters on an NMR spectra?

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15
Q

What is meant by the term chemical shift?

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16
Q

What scale is used to measure the chemical shift?

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17
Q

When do we get chemical shift equivalence? When is the chemical shift for a proton the same?

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18
Q

What are the chemical shift ranges, what type of functional groups do we find to the left (downfield) and right (upfield) on the chemical shift spectrum?

A

Think of it as electron density

Left (downfield) - Low electron density around nucleus - Presence of electron withdrawing group

Right (upfield) - high electron density around nucleus

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19
Q

What the hell can we actually use chemical shifts for?

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20
Q

What do we use integrals for/relative height of the peak? What happens to the integrals when the NMR reflects a mixture of molecules?

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21
Q

In NMR, what does coupling refer to?

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22
Q

What is scalar coupling?

Example - Two spin system?

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23
Q

What are the different possible scalar coupling patterns? What can they be used for?

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24
Q

Typical scalar coupling values?

A

Coupling constant (J) - Hz

Indicate the degree of splitting that is found between these interactions - to what extent the peaks split

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25
Q

What is one key difference that arises when performing NMR in an organic solvent vs. aq. solution?

A

Main difference - Exchangeable -OHs & -NH3 will only appear on the spectrum under specific conditions

  1. When NMR is performed in H2O –> OH shows no resonances due to rapid exchange with the high concentration of H2O (chemical shift averaged to H2O) unless exchange is slowed through internal H-bonding

Basically Protons attached to OH exposed to H2O will rapidly exchange - NMR can pick up on this - will be averaged out to H2O chemical shift

Likewise, NHs are similar to OH but are exchange at a slower rate at physiological pH - more readily observed

  1. When NMR is performed in Organic solvents - no free exchange of protons with these groups - Hence, we will get a peak for OHs and NHs (just like any other proton environments) + will contribute to the coupling (splitting) of peaks!

But!

If we were to add D2O to the organic solvent solution - we lose that proton signal plus any coupling it may have

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26
Q

Are NMR spectra normally recorded in H2O or organic solvents?

A

Normally NMR spectra are recorded in Water

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27
Q

What do these two graphs show you?

A

Remember…

T1 - Refers to the return towards the net bulk magnetization - equilibrium

T2 - Dispersal of spins –> entropic increase* - move away from spin coherence - increase in disorder

Answer from the overlord is pending! - Answered

28
Q

Why does the spin-spin relaxation change depending on the size of the molecule?

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29
Q

What does dipolar coupling refer to?

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30
Q

How is the Nuclear Nuclear Overhauser effect (NOE) used to indirectly measure dipolar coupling?

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31
Q

Why would we use Carbon-13 NMR?

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32
Q

Three type of Carbon-13 (13C) NMR Spectra we should be aware of?

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33
Q

Are the NMR spectra of peptides and proteins very complex

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34
Q

If an NMR spectra is very complex, how can we make it simpler to analyse?

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35
Q

How do we encode a second frequency dimension into our 2D NMR?

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36
Q

How would a 2D 1H-1H Correlation Spectroscopy (COSY) look like?

A
  1. Peaks that lie on the diagonal line correspond to the peaks one would see on a 1D NMR
  2. Cross peaks - They identify proton pairs that are in close proximity

COSY - Number of bonds away (Max 3-4)

NOESY - Less than 5Å away

37
Q

What do you mean by 2D NMR spectra can be used to resolve overlaps?

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38
Q

Draw a 2D COSY spectrum for the following molecule

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39
Q

Does each amino acid have its own COSY spectrum?

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40
Q

When drawing any sort of NMR spectra, whether that 1D, 2D etc., what should you include?

A

Apart from drawing the spectra out

YOU MUST include a written description to explain why you drew the spectra they way you did?

e. g.
1. What did you give that group that chemical shift - shielding?
2. What did you assign does scalar coupling patterns - peak splitting?
3. Why did you draw does cross-peaks on the 2D spectra?

41
Q

When would we observe a cross-peak between two environments on a NOESY spectra?

A
42
Q

Apart from COSY and NOESY, what other 2D spectra are there which we can use?

A
43
Q

Outline what a 13C-1H HMQC & 13C-1H HMBC are…

A
44
Q

Can you obtain integrals from a 2D NMR spectra?

A

2D spectra can be used to obtain integrals for example to quantify the number of carbons in HMQC and HMBC but..

It is more accurate to run a 1D spectra.

45
Q

On a 2D spectra are the frequencies on the X or Y axis recorded directly?

A

Remember we for a 2D spectra we encode one frequency into another

46
Q

Why do elementary (Protons, neutron, electron) particles have magnetic moment?

A

They possess charge + spin (angular momentum)

Hence, movement of a charged creates a magnetic field –> a magnetic moment is created

Thus, depending on the nuclear make of a nucleus we can get a magnetic moment

47
Q

Why does the proton have greatest magnetic moment?

A

Higher charged density results in greater magnetic moment

A proton is the smallest/highest charged density –> resulting in the greatest magnetic moment

48
Q

How do spin 1/2 particles, behave in an external magnetic field?

A

With spin ½ we have two orientations which means that they can align parallel or anti-parallel to th external magnetic field

Since it can’t align perfectly it processes around the external magnetic field

49
Q

How many different states does a particle with a spin quantum number equal to 1 have (I=1)?

A

Number of orientations - determined using the following equation 2I + 1

Where ‘I’ is the spin quantum number

Spin 1 has three orientations which are…

-1, 0 and +1

50
Q

What would happen if we were to increase the strength of the external magentic field?

A

Increase external magnetic field –> the energy difference between spin states increases

Hence, we use have to use a higher energy pulse to match this energy difference

51
Q

When running NMR, do you need highly concentrated solutions of your protein?

A

Yes, it is preferable to have highly concentrated solutions of your protein.

e.g. ~1mM or 15mg ml-1 for a 15 kDa protein

52
Q

What are the chemical shifts (rough estimate) that correspond to -CH3 compared to a aromatic group?

A
  1. -CH3 proton –> typically exhibits a chemical shift of 1ppm
  2. Aromatics proton –> typically exhibit a chemical shift of 7ppm

Note - Generally speaking the chemical shifts of protons range from 0 to 9 ppm!

53
Q

What size protein is NMR limited to?

A

Generally limited to proteins that are less than 50kDA - but the resolving power is certain to increase in the future

54
Q

What are the different scalar coupling patterns? How can we figure out the intensity ratio for each pattern?

A
55
Q

What is the N+1 rule - Peak splitting?

A
56
Q

How do you use the N+1 rule, when there are two hydrogen environments surrounding that are not equivalent, surrounding a central hyrdogen environment?

A

Apply the N+1 rule to each surrounding hydrogen environment and multiply them together - to get the splitting pattern

Notice that in this case we only have 6 peaks as some peaks overlap

But note….

You can either have

4 x 3 - Quartet of triplets

or…

3 x 4 - Triplet of quartets

57
Q

How can you draw the following NMR spectra…

4 x 3 - Quartet of triplets

or…

3 x 4 - Triplet of quartets

A
58
Q

Why do we get the intensity ratios that follow the pascals triangle?

A
59
Q

Explain why the following molecule would produce a doublet with a 1:1 ratio on the NMR spectra?

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60
Q

Why would the following molecule give rise to a triplet with an intensity ratio of 1:2:1?

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61
Q

Why would the following molecule give rise to a triplet with an intensity ratio of 1:3:3:1?

A
62
Q

How is intensity represented on 2D NMR spectra?

A

Intensity is depicted as countour lines - just like on a map that shows height

63
Q

How is 2D NMR data collected experimentally?

A
64
Q

Outline how to analyse HMQC/HSQC.

A
65
Q

Outline how to analyse HMBC?

A
66
Q

Why do we get signals on HMBC NMR spectra that don’t correspond to a H1 peak?

A

Known as satellite signals that lay symmetrically either side of a proton signal.

These signals are not a legitimate HMBC signal but rather HMQC signals bleading through on to our HMBC spectra - Show 1J Bonding

67
Q

On a HMBC, is there any correlation between 2J and 3J coupling and the signal strenght?

A

In Aromatic/conjugated systems

3J signal will almost always be stronger than 2J

In Aliphatic systems

2J tend to be stronger - but is more subjective

Imperial (Y2) - Structural Biology (6 decks)

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