3.5: NMR spectroscopy

Question 1

NMR: Atomic Nucleus

Answer 1

nuclei have magnet moment that comes from spin of protons and neutrons

> magnetic dipole moment

Nucleus can be depicted as bar magnet

Question 2

NMR: Principle

Answer 2

Nuclei = have magnetic moment

w/o field = no E diff
w/ field = E diff
> m = +1/2 m = -1/2

E diff
> corresponds to Radiowave frequency
> proportional to strength of the magnetic field

Question 3

NMR Layout

Answer 3

Superconducting magnets
+ Cooling
> provided by liq. nitrogen and helium

Sample Holder / Probe: Where the sample is placed inside the magnet.

RF Transmitter: Sends radio frequency pulses to excite nuclei.

RF Receiver: Detects emitted signals from the sample (Free Induction Decay).

Computer/Interface: Controls the experiment and processes data.

Fourier Transform + Display: Converts time-domain signal into spectrum for viewing.

Question 4

NMR Sample

Answer 4

high conc. in solution
> ensemble measurement

Question 5

NMR Isotope Problem

Answer 5

Nuclei with even protons and neutrons have spin = 0
> NMR - invisible

Magnetic moment/Relative NMR vary between isotopes also

Solution: Isotope Labelling

Question 6

NMR Isotope Labelling methods and pros/cons

Answer 6

1. Cell-free / In Vitro Translation
   > low yield, but allows specific labelling for large proteins too
2. Protein production in bacteria
   > Bottlenecked by size because complex proteins require eukaryotic folding which bacteria can’t do obv
   > high yield

Question 7

NMR Chemical shift

Answer 7

nuclear behaviour depends on local magnetic field
electrons are partially shielded
> chemical bonds influence local magnetic env.
> expressed in ppm

Question 8

NMR J-Coupling

Answer 8

Scalar coupling via bond-bond interactions
> splitting of energy levels

Question 9

NMR Nuclear Overhauser Effect (NOE)

Answer 9

Coupling by space
> via dipole-dipole interactions and spin polarisation
> Limited to scalar lengths of <0.5nm (1/r^6)

Question 10

NMR Spectra Info

Answer 10

• Line splitting / J-coupling
  > info through bond/neighbours
• Chemical shift position
  > structure
• Area under line
  > number of detected nuclei

Question 11

NMR: Larger proteins and solution

Answer 11

Spectra are crowded and overlap due to
> inc peak line width with inc molecular weight
> inc total number of peaks#

Solution: Require 2D visualisation

Question 12

2D-NOESY

Answer 12

H-H interactions that are closer than 5 Angstroms

Question 13

COSY

Answer 13

Hydrogen connected through chem bonds

Question 14

NMR: Protein structure determination steps

Answer 14

1. Isotope labelled protein sample
2. collect data
   > over weeks
3. peak assignment
4. collection of ‘restraints’ (nmr info)
   !!! 5. molecular dynamics simulation using force-field desc of protein and NMR restraints’ !!!

Question 15

NMR: Protein structure determination summary

Answer 15

• req. isotope labelled proteins
• in sol. ensemble measurement
• indirect structure determination from restraints
• limited by labelling and size of protein (<80kDa)

Allows:
diffusion rates, protein dynamics, relative domain orientations