BB7 Structures From NMR

Question 0

Nuclei with intrinsic magnetism

Answer 0

• 1-H
• 13-C
• 15-N
• 31-P

Question 1

Nuclear Magnetic Resonance (NMR)

Answer 1

• reveal atomic structures of macromolecules IN SOLUTION

Question 2

1-H can be used in NMR to see the structures of

Answer 2

proteins

• extensively found in biological systems

Question 3

31-P is used in NMR to study the structures of

Answer 3

nucleic acids

NOT PROTEINS

Question 4

The magnetism of nuclei comes from

Answer 4

the SPIN of their protons

Question 5

The spin of 1-H generates a

Answer 5

magnetic moment

Question 6

In an applied magnetic field, magnetic moment adopts 1 of 2 orientations

Answer 6

• α = orientated WITH the field

* β = orientated AGAINST the field

Question 7

B0 (sub)

Answer 7

applied magnetic field strength

Question 8

Delta E

Answer 8

• energy difference between α and β
• proportional to B0
• in the radio frequency range

Question 9

1-H nuclei in the α state can be excited into the β state

Answer 9

• requires pulse input of radio-frequency energy

Question 10

Pulse input - radio frequency

Answer 10

ν0 = γH0 / 2π

• ν0 = radio-frequency
• γ = magnetic ratio for a given nucleus

Question 11

Electrons around 1H

Answer 11

• alter the magnetism that the nucleus experiences
• creates a local chemical environment
• shields protons from the applied field

Question 12

Electrons shield protons from the applied field

Answer 12

H0 = B0 (1 - σ)

• H0 = local magnetic field strength
• σ = shielding factor (modifying agent)

Question 13

1H nuclei in different chemical environments will have different

Answer 13

ν0 values
• very small
• scaled as δ - ppm

Question 14

The local magnetic field is further altered through

Answer 14

• molecular bonds to neighboring nuclei with magnetic moments
• nuclei affect each other = spin-spin coupling

Question 15

Spin-spin coupling requirements

Answer 15

• neighboring nuclei must be in different chemical environments (even for seemingly identical groups)
• nuclei must be 3 or less bonds apart (otherwise too small to be seen)

Question 16

Relaxation

Answer 16

nuclei excited to the β state must lose energy to return to the α state
• occurs via interactions with other near neighbour magnetic nuclei

Question 17

An NRM spectrum is the observation of

Answer 17

β state protons falling back to the α state

Question 18

2 forms of relaxation

Answer 18

• relaxation through bonds

* relaxation through space

Question 19

Relaxation can also be observed by

Answer 19

• 2-D NMR spectroscopy

Question 20

2-D NRM spectroscopy

Answer 20

• . spectra are drawn as a 2-D contour map of peaks
• peaks on the diagonal are the 1-D NMR spectra
• peaks off the diagonal show where relaxation interactions have occurred

Question 21

2-D correlation spectroscopy (COSY)

Answer 21

• gives structural info through bond connections between residues
• identifies the residues

Question 22

2-D nuclear overhauser spectroscopy (NOESY)

Answer 22

• through space or bond effect
• reveals info about structure through space relaxation interactions
• uses Nuclear Overhauser Effect (NOE) data
• peaks on diagonal spectrum are a normal 1-D spectrum
• used as markers to locate the off-diagonal peaks
• 1H nuclei close space to each other will share an off-diagonal peak (symmetric in appearance)
• off-diagonal peaks shower where relaxation has occurred between 1H nuclei through space
• large number of off-diagonal peaks are present in a 2-D spectrum (represent nuclei in close proximity to each other)

Question 23

Ensemble of structures

Answer 23

NOESY together with additional data obtainable from an NMR , a SET of solution conformation of a protein can be obtained
• near terminal
• through bond effects, not space
• side chains on extremes, floppy

Question 24

Potential problems of NOESY

Answer 24

* no SINGLE structure exists for a protein - NOESY data for a range of lengths * methyl groups often dealt with as a single atom - pseudo atoms