1. Biomolecular Nuclear Magnetic Resonance Spectroscopy
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”

2. 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)

3. Biomolecular NMR: primarily spin 1/2 nuclei (1H, 13C, 15N, 31P)
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)

4. Nuclei With Non-Zero Spin Align in Magnetic Fields
DE = h g Ho
Efficiency factor-
nucleus
Constants
Strength of
magnet Ho
Alignment
Energy
parallel
anti-parallel

5. NMR: The Bar Magnet Analogyp p p ap ap ap + - + - + - + - - + - + + -
Ho = - + - + - + - + - + - + + - + - + -
1. force non-alignment
2. release

6. Resonance: Perturb Equilibriumap
1. equilibrium DE
DE = h g Ho
Efficiency factor-
nucleus
Constants
Strength of
magnet Ho
hn = DE H1
2. pump in energy p ap
3. non-equilibrium

7. Return to Equilibrium (Relax): Read Out Signalsp ap DE
3. Non-equilibrium
hn = DE
4. release energy (detect) p ap
5. equilibrium

8. Magnetic Resonance SensitivityNp
Nap
= e
-DE/kT
Sensitivity (S) ~ D(population)
S ~ DN =
DE = h g Ho
Efficiency factor-
nucleus
Constants
Strength of
magnet
DE is small
At room temp., DN ~ 1:105
Intrinsically low sensitivity
Need lots of sample
Increase sensitivity by increasing magnetic field strength

9. Intrinsic Sensitivity of Nuclei
Nucleus g % Natural Relative
Abundance Sensitivity
1H 2.7 x
13C 6.7 x
15N x
31P 1.1 x

10. The Classical Treatment: Nuclear Spin Angular Momentum
Two spins
All spins  Sum
Bulk Magnetization
excess
facing
down Ho
parallel
anti-parallel
Torque + int. motion = precession
Precession around Z axis
Larmor frequency ():
DE = hgHo  DE = hn  n = H0 = 

11. Pulse Fourier Transform NMR
90ºx RF pulse =
 =  H0 Ho Ho A t
Fourier
Transform f 
NMR frequency
Variation of signal at X axis vs. time

12. The Power of Fourier Transform
90ºx RF pulse +
1 =  H0
2 =  H0 A t
Fourier
Transform f 2 1
NMR frequency domain
Spectrum of frequencies
NMR time domain
Variation in amplitude vs time

13. The Pulse FT NMR Experiment
equilibration
90º pulse
detection of signals
Experiment
(t)
Data
Analysis
Fourier
Transform
Time domain (t)

14. NMR Terminology Chemical Shift & Linewidth
The exact resonance frequency (chemical shift) is determined by the electronic environment of the nucleus

15. NMR Scalar and Dipolar Coupling
Through
Space
Through
Bonds
Coupling of nuclei gives information on structure

16. Resonance Assignment
The key attribute: use the scalar and dipolar couplings to match the set of signals with the molecular structure
CH3-CH2-OH OH
CH2
CH3
Which signal from which H atoms?

17. Proteins Have Too Many Signals! 1H NMR Spectrum of Ubiquitin
~500 resonances
Resolve resonances by multi-dimensional experiments