NMR Spectoscopy

Question 1

Dynamic processes which NMR helps to study

Answer 1

• Protein folding
• Protein hydration
• Ligand binding
• Enzyme kinetics

Question 2

Protein-Ligand Interaction

Answer 2

• If there is binding of the ligand to the protein, some peaks will change, larger the change, closer the ligand is to the corresponding nucleus
  in space

Question 3

Basic Principle of NMR Spectroscopy

Answer 3

Question 4

Nuclear Spin State (I)

Answer 4

Isotopically labelling, by growing proteins in culture with C13 and N15. Using probes

Question 5

Modern NMR Spectrometers

Answer 5

Built to observe nuclear “spin
flips” – all consist of a magnet, a source of radiofrequency and a
probe to accommodate the sample

Question 6

Basic Principle

Answer 6

Nuclei lined up and hit with 90 degree radiofrequency pulse
Acquisition Phase
tD = Recovery time

Question 7

Example (CH3CH2OH)

Answer 7

Question 8

What information does 1H NMR Spectroscopy give us?

Answer 8

1) Chemical Shift - Indicating nature of functional groups

2) How many protons present in each chemical environment => molecular symmetry

3) Peak Splitting - relationships of groups of protons to one another

Question 9

Chemical Shift

Answer 9

Question 10

Effect of greater shielding on position of resonance

Answer 10

Question 11

Effect of less shielding on position of resonance

Answer 11

Question 12

Shielding Effects

Answer 12

Question 13

1H NMR Chemical Shifts in Alkenes (double bonds)

Answer 13

Question 14

Chemical Shift Anisotropy

Answer 14

= Bonds create anisotropic chemical shifts in molecules
- Shielded and deshielded regions

Question 15

Summary of Chemical Shifts

Answer 15

Question 16

Integration

Answer 16

Question 17

Peak Splitting / Coupling

Answer 17

Question 18

2D NMR (COSY)

Answer 18

Require NMR experiments that consist of several pulses of radiofrequency radiation, together with variable delays between these pulses

Question 19

Adv of NMR

Answer 19

• Generally applicable for obtaining a complete set of 3D- information (primary, secondary and tertiary structure) for most small/medium-sized proteins
• Uniquely able to directly study dynamic/functional aspects of
  proteins (e.g. catalysis, receptor binding, folding etc.
• Offers the possibility to study dynamic properties that occur in
  solution, just as they would in the living cell

Question 20

Comparison of Common Techniques used in Protein Structure Determination

Answer 20

Question 21

Requirements for Protein

Answer 21

• 1-10 mg sample
• Close to 100% purity as possible
• Sample must be soluble in 90% H2O/10% D2O
• pH 7 or less to prevent Hs exchanging
• Stable over period of days
• Paramagnetic ions avoided
• Unlabelled proteins ~ 10kDa
• C13 N15 labelled proteins ~ 30 kDa

Question 22

NMR of unlabelled proteins

Answer 22

TOCSY extends the reach of COSY into sidechains

Question 23

Polypeptides/Protein from NMR viewpoint

Answer 23

Question 24

20 AAs classified for NMR

Answer 24

Question 25

NOESY

Answer 25

Detect whether protons are close to one another in space
- Determine the position of an amino acid in the sequence (primary
structure)
- Determine the three-dimensional shape of the protein (secondary and
tertiary structure)
- The size of the nOe can be calculated from a two-dimensional
volume integral
- Establishes the order of AAs in primary stucture

Question 26

General conclusion for the applicability of of COSY/TOCSY & NOESY strategy for larger proteins

Answer 26

Likely to become less and less successful in assigning resonances in the backbone as proteins become larger. The more amino acids there
are in the molecule, the higher the probability of overlap between 1H
resonances in the protein backbone

Question 27

3D NMR Techniques

Answer 27

Double-labelled proteins

Question 28

High Field NMR Spectroscopy & Current uses

Answer 28

• Higher magnetic strength, better quality structures
• Macromolecular complexes and other ‘omics’