Biomolecular Nuclear Magnetic Resonance Spectroscopy

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Biomolecular Nuclear Magnetic Resonance
Spectroscopy
02/02/09

• 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
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Nuclear Spin

• Nuclear spin angular momentum is a quantized
  property of the nucleus in each atom
• The nuclear spin angular momentum of each atom is
  represented by a nuclear spin quantum number (I)
• All nuclei with even mass numbers have I0,1,2
• All nuclei with odd mass numbers have
  I1/2,3/2...
• NMR is possible with all nuclei except I0 (e.g.
  12C), but I1/2 has simplest spin physics
• Biomolecular NMR ? primarily 1H, 13C, 15N (31P)

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Spin 1/2 Nuclei in a Magnetic Field
Bo
Energy
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Intrinsic 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
Prepare samples enriched in these nuclei
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Variables Affecting Sensitivity
- DE is very small ? DN is small - DN 1105
(at room T) NMR has low sensitivity ? requires
lots of sample!
Increase sensitivity by increasing magnetic field
strength or reducing electronic noise
(cryo-probes)
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The Resonance Experiment
Strength of signal ? D (population)
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NMR TerminologyChemical Shift Linewidth
The exact resonance frequency (chemical shift) is
determined by the electronic environment of the
nucleus
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Scalar and Dipolar Coupling
Through Bonds
Through Space

• Coupling of nuclei gives information on structure

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Resonance Assignment
CH3-CH2-OH
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2D NMR Spectra FacilitateIdentification of
Coupling
Coupled spins
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Biomolecules Have Many Signals
1H NMR Spectrum of Ubiquitin 75 residues, 500
1H resonances

• Terminology signals are overlapped

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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

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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

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Regions of the 1H NMR Spectrumand Dispersion by
the 3D Fold
What would the unfolded protein look like?
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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

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Spectra of BiomacromoleculesOverlapped
Sub-Spectra
Groups of coupled nuclei Each residue in the
sequence gives rise to an independent NMR
sub-spectrum, which makes the problems much
simpler than if all spins were coupled
Methods have been developed to extract each
sub-spectrum from the whole
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Strategy to AssignResonances in a Protein

1. Identify resonances for each residue (scalar)
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)
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Even Sub-Spectra are Overlapped!
1H NMR Spectrum of Ubiquitin

• Resolve resonances by multi-dimensional
  experiments

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Use 2D NMR to Resolve Overlapping Signals
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Multi-Dimensional NMR
If 2D cross peaks overlap? go to 3D or 4D ..
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Another Solution tothe Overlap Challenge

1. Increase dimensionality of spectra to better
   resolve signals 1D?2D?3D?4D.
2. Use hetero nuclei (13C,15N) to distinguish,
   coupling is efficient because only 1-bond

Hz
HA
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Multi-Dimensional Heteronuclear NMR
15N-1H HSQC
F1 Chemical Shift (15N)
F2 Chemical Shift (1H)
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Advantages ofHeteronuclear nD NMR
Uses a second nucleus to resolve overlap of the
first chemical shift of each nucleus is
sensitive to different factors More information
to identify resonances Less sensitive to MW
because this strategy uses large 1 and 2-bond
scalar couplings