Transcript Center for Structural Biology

01/15/03
Biomolecular Nuclear Magnetic
Resonance Spectroscopy
BASIC CONCEPTS OF NMR
• How does NMR work?
• Pulse FT NMR
• 2D NMR experiments
• nD NMR experiments
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
2D NMR: Coupling is the Key
2D detect signals twice
(before/after coupling)
90º pulse
t1
Same as 1D
experiment
Transfers between
coupled spins
2D NMR Pulse Sequence
t2
t1
t2
2D NMR Spectrum
Pulse Sequence
t1
Spectrum
Before mixing
After mixing
Coupled spins
t2
The Power of 2D NMR:
Resolving Overlapping Signals
1D
2 signals
overlapped
2D
2 cross peaks
resolved
Acronyms For Basic Experiments
Differ Only By The Nature Of Mixing
Scalar Coupling
Dipolar Coupling
Homonuclear
Heteronuclear
COSY
HSQC
TOCSY
Hetero-TOCSY
Multiple
Quantum
HMQC
NOESY
NOESY-HSQC
NOESY-HMQC
Multi-Dimensional NMR:
Built on the 2D Principle
3D- detect signals 3 times
90º pulse
(t3)
t1
Same as 1D
experiment
t2
t3
3D NMR Pulse Sequence
Experiments are composites  acronyms are composites