Structural Biology

2nd year

Biological NMR spectroscopy

Professor Steve Matthews

 Main applications of NMR
• Analytical – in synthesis for quickly checking for correct product
• Chemical structure – Chemical shifts and couplings for indentifying molecular
structure
• 3D structure determination –nuclear Overhauser effects (NOEs) for measuring
distances
• Molecular interactions – chemical shift mapping and NOEs in protein
interactions
• Molecular dynamics – relaxation and the NMR time scale for monitor the
kinetics of processes
• NMR imaging – encoding spatial dimensions in data
• Metabolomics – metabolic profiling of organisms
Objectives:
• A working knowledge of nuclear magnetic resonance (NMR)

• An understanding of basic NMR experiments

• The ability to interpret simple 1D and 2D spectra

Contents:
L1 -- Introduction to the principles of NMR

L2 – NMR parameters and Interpret 1H 1D spectra

L3 – 2D 13C/1H NMR and approaches for proteins

L1 – Introduction to the principles of
NMR
 What is nuclear spin?
• Elementary particles possess an intrinsic angular momentum, I,
known as spin
• Spin is quantised and comes in multiples of ½ such that there are
(2I + 1) values between –I and +I (I is spin quantum number)
• Protons, unpaired electrons (EPR/ESR) and neutrons possess spin
and I = ½
• Nuclei comprising even protons and neutrons have 0 spin
angular momentum. Nuclei with odd proton and neutrons
have integral spin quantum numbers. The rest have
1/2-integral spin.
• A charged nucleus rotating with angular
frequency creates a magnetic field B
 Spin energy levels
Bo
b (mI= -1/2)
Planks law
DE = h𝛖
then
DE radiofrequency
𝛖 = gBo/2π Hz
DE = 𝛄ħBo
a (mI=1/2)
𝛄 is magnetogyric ratio)
(

• Strength of applied magnetic field determines the energy

gap of a given nuclei
• Higher magnetic fields give higher sensitivity, but..
• Energy gap 10-5 that of infrared
 Frequently used nuclei with spin
Nuclei Spin Abundance % γ MHz/T Sensitivity
1
H ½ ~100 42.58 1
31
P ½ 100 17.25 0.0663
13
C ½ 1.1 10.71 0.000176
15
N ½ 0.025 4.32 0.0000039
2
H(D) 1 1 6.54 0.0000015

• The proton (1H) is the most sensitive nucleus

• 13C/15N are low abundance and therefore low sensitivity
• The deuteron (2H) is NMR active
 Recording an NMR spectrum
Radiofrequency can be swept
b (mI= -1/2)
through all resonances and the
absorption is measured

DE 1st Proton NMR spectrum

paraffin wax
(Bloch and Purcell - 1945)
a (mI=1/2)

This is very inefficient - Can we do better?

 Larmor precession of nuclear spins
The magnetic
moment (μ) associated with a
spinning spherical charge will Precession
precess in an external
μ
magnetic field (B0)
B0 Spin rotation
Known as the Larmor
precession.

Absorption occurs when the

radiofrequency matches the
Larmor frequency.
 Macroscopic magnetisation of a sample
Large numbers of spin 1/2 nuclei at equilibrium in a strong external
magnetic field (Bo).

z
ω0
z Net magnetization (M0)
x Summed
x
over
y ensemble
y
 The effect of an radiofrequency pulse
z z z z

x x x
y y y

Radiofrequency
pulse
Net magnetisation shifts away from the z-axis and toward the y-axis:
1. This is due to spin flips between between +1/2 and -1/2 states
until, and
2. The spin precessions about the z-axis becomes coherent (non-
random), generating a significant y component to the net
magnetization (M).
 A short radiofrequency pulse
Fourier pairs are two functions
A short radio frequency
pulse contains many
frequencies in a broad
band and thus can excite
resonances of all nuclear
spins in a sample at the
same time.

Time domain Frequency domain

Note: think of how you construct a truncated wave

Pulsed Fourier transform NMR
Decaying radiofrequency
signal generated in the
receiver coil Is called the
‘free induction decay’ (FID)

Related to the traditional

frequency spectrum though
a Fourier Transform