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01/28/08
Biomolecular Nuclear Magnetic
Resonance Spectroscopy
BASIC CONCEPTS OF NMR
• How does NMR work?
• Resonance assignment
• Structural parameters
Reading: Chapter 22 in Protein and Peptide Drug Analysis
“Solution Structure Determination of Proteins by NMR”
Nuclear Spin
• Nuclear spin angular momentum is a quantized
property of the nucleus in each atom, which arises from
the sub-atomic properties of neutrons and protons
• The nuclear spin angular momentum of each atom is
represented by a nuclear spin quantum number (I)
• All nuclei with even mass numbers have I=0,1,2…
• All nuclei with odd mass numbers have I=1/2,3/2...
• NMR is possible with all nuclei except I=0, but I=1/2 has
simplest physics
Biomolecular NMR  primarily 1H, 13C, 15N (31P)
Spin 1/2 Nuclei in a Magnetic Field
Efficiency factornucleus
Bo
Energy
DE = h g Bo
Constants
Strength of
magnet
Intrinsic Sensitivity of Nuclei
Nucleus
g
% Natural
Abundance
Relative
Sensitivity
1H
2.7 x 108
99.98
13C
6.7 x 107
1.11
0.004
15N
-2.7 x 107
0.36
0.0004
31P
1.1 x 108
100
Prepare samples enriched in these nuclei
1.0
0.5
Variables Affecting Sensitivity
Sensitivity (S) ~ D pop. (N vs N)
S ~ DN =
N
= e-DE/kT
N
- DE is very small  DN is small
- DN ~ 1:105 (at room T)
NMR has low sensitivity 
requires lots of sample!
Efficiency factornucleus
DE = h g Ho
Constants
Strength of
magnet
Increase sensitivity by increasing magnetic field strength
or reducing electronic noise (cryo-probes)
The Resonance Experiment
DE
Bo
B1
hn = DE
Equilibrium
Pump in energy
(RF transmitter)
Non-equilibrium
Strength of signal
 D (population)
NMR signals
hn = DE
Release energy
(RF receiver)
Equilibrium
NMR Terminology
Chemical Shift & Linewidth
The exact resonance frequency (chemical shift) is
determined by the electronic environment of the nucleus
Scalar and Dipolar Coupling
Through
Space
Through
Bonds
 Coupling of nuclei gives information on structure
Resonance Assignment
CH3-CH2-OH
OH
CH2 CH3
Which signal from which H atoms?
The key attribute: use the scalar and dipolar couplings to
match the set of signals with the molecular structure
2D NMR Spectra Facilitate
Identification of Coupling
F1
Coupled spins
F2
Biomolecules Have Many Signals
1H
NMR Spectrum of Ubiquitin
~75 residues, ~500 1H resonances
Terminology: signals are overlapped
Challenges For Using NMR to
Study Biological Macromolecules
• Hundreds-thousands of signals!
• Must assign the specific signal for each
atom
• Thousands of couplings between
nuclei- these also need to be assigned
Critical Features of
Protein NMR Spectra
• Regions of the spectrum correspond to
different parts of the amino acid
• Tertiary structure leads to increased
dispersion of resonances
Regions of the 1H NMR Spectrum
and Dispersion by the 3D Fold
What would the unfolded protein look like?
Critical Features of NMR Spectra
of Biomolecules
• Regions of the spectrum correspond to
different parts of the amino acid
• Tertiary structure leads to increased
dispersion of resonances
• Bio-macromolecules are polymers 
The nuclei are coupled to some (but not
all!) other nuclei
Spectra of Biomacromolecules:
Overlapped Sub-Spectra
*Groups of coupled nuclei*
Each residue in the sequence gives rise to an
independent NMR sub-spectrum, which is much
simpler than the complete spectrum
Methods have been
developed to extract
each sub-spectrum
from the whole
Strategy to Assign
Resonances in a Protein
1. Identify resonances for each residue
(scalar)
T G L S
R G
S
2. Put amino acids in order (dipolar)
1
2
3
4
5
6
7
R-G-S-T-L-G-S
Same idea for any biopolymer (e.g. DNA, RNA)
Even Sub-Spectra are Overlapped!
1H
NMR Spectrum of Ubiquitin
 Resolve resonances by multi-dimensional experiments
Use 2D NMR to Resolve
Overlapping Signals
1D
Sub-spectra
overlapped
2D
Crosspeaks
resolved!
Coupled spins
Multi-Dimensional NMR
Hb
Ha
HN
If 2D cross peaks
overlap go to 3D
or 4D …..
Another Solution to
the Overlap Challenge
1. Increase dimensionality of spectra to better
resolve signals: 1D2D3D4D….
2. Use attached nuclei (13C,15N) to distinguish
t2
Hz
HA
t1
t3
Multi-Dimensional Heteronuclear NMR
15N-1H
HSQC
F1 Chemical Shift
(15N)
F2 Chemical Shift
(1H)
Advantages of
Heteronuclear nD NMR
Uses a second nucleus to resolve overlap of the
first: chemical shift of each nucleus is
characteristic/sensitive to different factors
More information to identify resonances
Less sensitive to MW because this strategy uses
large 1 and 2-bond scalar couplings
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