Download Center for Structural Biology

01/23/04
Biomolecular Nuclear Magnetic
Resonance Spectroscopy
RESONANCE ASSIGNMENT IN PROTEINS
• Multi-dimensional NMR experiments
• NMR analysis of proteins
Proteins Have Too Many Signals!
1H
NMR Spectrum of Ubiquitin
~500 resonances
Resolve resonances by multi-dimensional experiments
The Pulse FT NMR Experiment
90º pulse
Experiment
(t)
equilibration
detection of signals
Fourier
Transform
Data
Analysis
Time domain (t)
The 2D NMR Pulse Sequence
+
1D + 1D = 2D
[2nd preparation]
2D NMR: Coupling is the Key
2D detect signals twice
(before/after coupling)
90º pulse
t1
Same as 1D
experiment
Transfers between
coupled spins
t2
t1
t2
The 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
Higher 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
Proteins Have Too Many Signals!
1H
NMR Spectrum of Ubiquitin
~500 resonances
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
NMR Spectrum to 3D Structure
H
H
H
H
H H H
H
H
H
H
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
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
Regions of the
1H
NMR Spectrum
What would the unfolded protein look like?
Solutions to the Challenges
1. Increase dimensionality of spectra to
better resolve signals: 123 4
t2
t1
t3
Resolve Peaks By Multi-D NMR
A BONUSregions in
2D spectra provide
protein fingerprints
If 2D cross peaks
overlap go to 3D
Solutions to the Challenges
1. Increase dimensionality of spectra to
better resolve signals: 123 4
2. 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
Double-Resonance Experiments
Increases Resolution/Information Content
15N-1H
R
R
-15N - Ca- CO -15N - Ca
H
H
HSQC
Large Scalar Couplings  Less
Sensitive to MW of the Protein
 Superior to 1H
homonuclear NMR:
all JH-H < 20 Hz
 Mixing is faster so
less time for signal to
relax