Biomolecular Nuclear Magnetic Resonance Spectroscopy

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Biomolecular Nuclear Magnetic Resonance
Spectroscopy
01/17/03

• RESONANCE ASSIGNMENT IN PROTEINS
• Multi-dimensional NMR experiments
• NMR analysis of proteins

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Proteins Have Too Many Signals! 1H NMR Spectrum
of Ubiquitin
500 resonances

• Resolve resonances by multi-dimensional
  experiments

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The Pulse FT NMR Experiment
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2D NMR Coupling is the Key
2D detect signals twice (before/after coupling)
90º pulse
Transfers between coupled spins
Same as 1D experiment
2D NMR Pulse Sequence
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The 2D NMR Spectrum
Pulse Sequence
t1
t2
Spectrum
Before mixing
Coupled spins
After mixing
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The Power of 2D NMRResolving Overlapping Signals
1D
2 signals overlapped
2D
2 cross peaks resolved
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Higher Dimensional NMRBuilt on the 2D Principle
3D- detect signals 3 times
90º pulse
(t3)
Same as 1D experiment
3D NMR Pulse Sequence

• Experiments are composites ? acronyms are
  composites

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Acronyms For Basic ExperimentsDiffer Only By The
Nature Of Mixing
Homonuclear
Heteronuclear
Scalar Coupling
COSY
HSQC
TOCSY
Hetero-TOCSY
HMQC
Multiple Quantum
Dipolar Coupling
NOESY
NOESY-HSQC
NOESY-HMQC
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Proteins Have Too Many Signals! 1H NMR Spectrum
of Ubiquitin
500 resonances
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Challenges For Determining Protein Structures
Using NMR

• Proteins have thousands of signals
• Assign the specific signal for each atom
• Thousands of interactions between atoms- also
  need to be assigned
• Need to transform representation from spectrum
  through interactions between atoms to spatial
  coordinates

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NMR Spectrum to 3D Structure
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Critical Features of Protein NMR Spectra

• The nuclei are not all mutually coupled
• Each amino acid gives rise to an independent NMR
  sub-spectrum, which is much simpler than the
  complete protein spectrum

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Critical Features of Protein NMR Spectra

• The nuclei are not all mutually coupled
• 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 Spectrum
What would the unfolded protein look like?
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Solutions to the Challenges

1. Increase dimensionality of spectra to better
   resolve signals 1?2?3 ?4

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Resolve Peaks By Multi-D NMR
A BONUS?regions in 2D spectra provide protein
fingerprints
If 2D cross peaks overlap? go to 3D
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Solutions to the Challenges

• Increase dimensionality of spectra to better
  resolve signals 1?2?3 ?4
• Detect signals from heteronuclei (13C,15N)
• Better resolution of signals/chemical shifts not
  correlated between nuclei
• More information to identify signals
• Lower sensitivity to MW of protein

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Double-Resonance ExperimentsIncreases
Resolution/Information Content
15N-1H HSQC
-15N - Ca- CO -15N - Ca
H
H
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Large Scalar Couplings ? Less Sensitive to MW of
the Protein

• Superior to 1H homonuclear NMR H-H couplings
  lt20 Hz
• Mixing is faster so less time for signal to relax