Download Center for Structural Biology - Structural Biology @ Vanderbilt

01/21/04
Biomolecular Nuclear Magnetic
Resonance Spectroscopy
BASIC CONCEPTS OF NMR
• How does NMR work?
• Pulse FT NMR
• Resonance assignment
NMR text: Chapter 22 in Protein and Peptide Drug Analysis
“Solution Structure Determination of Proteins by NMR”
NMR in Medicine and Biology
• MRI- Magnetic Resonance Imaging (water)
• In-vivo spectroscopy (metabolites)
• Solid-state NMR (large structures)
• Solution NMR
– Bioanalytical, primary structure
– Three-dimensional structure
– Molecular motions
– Molecular interactions- binding, reactions
Ligand screening (Pharma)
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 odd mass numbers have I=1/2,3/2...
• Nuclei with even mass numbers and an even number of
protons have I=0
• Nuclei with even mass numbers and an odd number of
protons have I=1,2,3…
Biomolecular NMR: primarily spin 1/2 nuclei (1H, 13C, 15N, 31P)
Nuclei With Non-Zero Spin
Align in Magnetic Fields
Efficiency factornucleus
Ho
Alignment
parallel
anti-parallel
Energy
DE = h g Ho
Constants
Strength of
magnet
NMR: The Bar Magnet Analogy
p
p
ap
ap
ap
+
-
+
-
+
-
1. force non-alignment
+
-
+
-
+
-
-
+
-
=
+
-
+
-
Ho
+
-
+
+
-
2. release
+
-
+
-
+
-
+
-
p
Resonance: Perturb Equilibrium
p
Ho
DE
1. equilibrium
Efficiency factornucleus
ap
H1
hn = DE
DE = h g Ho
2. pump in energy
Constants
p
ap
3. non-equilibrium
Strength of
magnet
Return to Equilibrium (Relax):
Read Out Signals
p
DE
3. Non-equilibrium
ap
hn = DE
4. release energy (detect)
p
5. equilibrium
ap
Magnetic Resonance Sensitivity
Sensitivity (S) ~ D(population)
S ~ DN =
Np
= e-DE/kT
Nap
DE is small
At room temp., DN ~ 1:105
Intrinsically low sensitivity
Need lots of sample
Efficiency factornucleus
DE = h g Ho
Constants
Strength of
magnet
Increase sensitivity by increasing magnetic field strength
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.
1.0
0.5
The Classical Treatment:
Nuclear Spin Angular Momentum
Two spins
All spins
 Sum
Ho
parallel
anti-parallel
Torque + int. motion = precession
Precession around Z axis
Larmor frequency ():
DE = hgHo  DE = hn  n = gH0 = 
excess
facing
down
Bulk
Magnetization
Pulse Fourier Transform NMR
t
90ºx RF pulse
=
 = g H0
Ho
Ho
A
t
f

NMR frequency
Fourier
Transform
Variation of signal
at X axis vs. time
The Power of Fourier Transform
t
90ºx RF pulse
+
1 = g H0
2 = g H0
A
t
f
2 1
Fourier
Transform
NMR frequency domain
NMR time domain
 Spectrum of frequencies
 Variation in amplitude vs time
The Pulse FT NMR Experiment
90º pulse
Experiment
(t)
equilibration
detection of signals
Fourier
Transform
Data
Analysis
Time domain (t)
NMR Terminology
Chemical Shift & Linewidth
The exact resonance frequency (chemical shift) is
determined by the electronic environment of the nucleus
NMR 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
Proteins Have Too Many Signals!
1H
NMR Spectrum of Ubiquitin
~500 resonances
Resolve resonances by multi-dimensional experiments