Title: Biomolecular Nuclear Magnetic Resonance Spectroscopy
1Biomolecular Nuclear Magnetic Resonance
Spectroscopy
01/21/04
- 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
2NMR 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)
3Nuclear 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
I1/2,3/2... - Nuclei with even mass numbers and an even number
of protons have I0 - Nuclei with even mass numbers and an odd number
of protons have I1,2,3 - Biomolecular NMR primarily spin 1/2 nuclei (1H,
13C, 15N, 31P)
4Nuclei With Non-Zero SpinAlign in Magnetic Fields
Ho
Alignment
Energy
parallel
anti-parallel
5NMR The Bar Magnet Analogy
p
p
p
ap
ap
ap
-
-
-
Ho
-
1. force non-alignment
2. release
6Resonance Perturb Equilibrium
Ho
7Return to Equilibrium (Relax) Read Out Signals
8Magnetic Resonance Sensitivity
DE is small At room temp., DN
1105 Intrinsically low sensitivity? Need lots of
sample
Increase sensitivity by increasing magnetic field
strength
9Intrinsic Sensitivity of Nuclei
Nucleus g Natural Relative
Abundance Sensitivity 1H 2.7 x 108
99.98 1.0 13C 6.7 x 107
1.11 0.004 15N -2.7 x 107
0.36 0.0004 31P 1.1 x 108
100. 0.5
10The Classical TreatmentNuclear Spin Angular
Momentum
Ho
parallel
anti-parallel
- Torque int. motion precession
- Precession around Z axis
- Larmor frequency (?)
- DE hgHo ? DE hn ? n ?H0 ?
11Pulse Fourier Transform NMR
t
90ºx RF pulse
? ? H0
Ho
Ho
A
t
Fourier Transform
f
?
Variation of signal at X axis vs. time
NMR frequency
12The Power of Fourier Transform
t
90ºx RF pulse
?1 ? H0 ?2 ? H0
A
t
Fourier Transform
f
?1
?2
- NMR frequency domain
- Spectrum of frequencies
- NMR time domain
- Variation in amplitude vs time
13The Pulse FT NMR Experiment
14NMR TerminologyChemical Shift Linewidth
The exact resonance frequency (chemical shift) is
determined by the electronic environment of the
nucleus
15NMR Scalar and Dipolar Coupling
Through Bonds
Through Space
- Coupling of nuclei gives information on structure
16Resonance Assignment
CH3-CH2-OH
Which signal from which H atoms?
17Proteins Have Too Many Signals! 1H NMR Spectrum
of Ubiquitin
500 resonances
- Resolve resonances by multi-dimensional
experiments