Biomolecular Nuclear Magnetic Resonance Spectroscopy
BASIC CONCEPTS OF NMR
• How does NMR work?
• Resonance assignment
• Structural parameters
02/03/10
Reading: Chapter 22 in Protein and Peptide Drug Analysis“Solution Structure Determination of Proteins by NMR”
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 I=0,1,2…
• All nuclei with odd mass numbers have I=1/2,3/2...
• NMR is possible with all nuclei except I=0 (e.g. 12C), but I=1/2 has simplest spin physics
Biomolecular NMR primarily 1H, 13C, 15N (31P)
Spin 1/2 Nuclei in a Magnetic Field
Bo Energy
E = h Bo
Nuclearefficiency factor:
(gyro-magnetic ratio)
Constants Strength ofmagnet
Intrinsic Sensitivity of Nuclei
Nucleus 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
Variables Affecting Sensitivity
-E is very small N is small - N ~ 1:105 (at room T)
NMR has low sensitivity NMR has low sensitivity requires lots of sample!requires lots of sample!
E = h Ho
Efficiency factor-nucleus
Constants Strength ofmagnet
N
N= e-E/kT
Sensitivity (S) ~ pop. (N vs N)
S ~ N =
Increase sensitivity by increasing magnetic field strength Increase sensitivity by increasing magnetic field strength or reducing electronic noise (cryo-probes)or reducing electronic noise (cryo-probes)
The Resonance Experiment
Bo EquilibriumE
hEB1 Pump in energy
(RF transmitter)
Non-equilibrium
Equilibrium
hE Release energy (RF receiver)
NMR signals
Strength of signal (population)
NMR TerminologyChemical Shift & Linewidth
The exact resonance frequency (chemical shift) is determined by the electronic environment of the nucleus
Scalar and Dipolar Coupling
Coupling of nuclei gives information on structure
ThroughBonds
ThroughSpace
Resonance Assignment
CH3-CH2-OH
Which signal from which H atoms?
OH CH2 CH3
Approach: use the scalar and dipolar couplings to match the set of signals with the molecular structure
2D NMR Spectroscopy FacilitatesIdentification of Coupling
Partners
Coupled spins
F1 F2
Biomolecules Have Many Signals
1H NMR Spectrum of Ubiquitin~75 residues, ~500 1H resonances
Terminology: signals are overlapped
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
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
Regions of the 1H NMR Spectrumand Dispersion by the 3D Fold
What would the unfolded protein look like?
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
Spectra of Biomacromolecules:Overlapped 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 to all other spins
Methods have been developed to extract each sub-spectrum from the whole
Basic Strategy to AssignResonances in a Protein
1. Identify resonances for each residue (scalar)
2. Put residues in order (dipolar, scalar)
1 2 3 4 5 6 7
R - G - S - T - L - G - S
LT G S S R G
Same idea for any biopolymer (e.g. DNA, RNA)
Even Sub-Spectra are Overlapped!
Resolve resonances by multi-dimensional experiments
1H NMR Spectrum of Ubiquitin
Solutions to the Overlapof Sub-spectra
1. Increase dimensionality of spectra to better resolve signals: 1D2D3D4D….
Use of 2D NMR to Resolve Overlapping Sub-spectra in 1D
1D Sub-spectraoverlapped
2D
Coupled spins
Off-diagonalcross peaks
resolved!
Overlap in 2D NMR Spectra
If 2D cross peaks overlap go to 3D or 4D or …..
Solutions to the Challenge of Overlap in nD NMR Spectra
1. Increase dimensionality of spectra to better resolve signals: 1D2D3D4D….
2. Use hetero nuclei (13C,15N) to distinguish 1H cross peaks
t2
t1
t3HA
Hz
2D Heteronuclear NMR Spectroscopy
15N-1H HSQC
F1
Ch
emic
al S
hif
t(15
N)
F2 Chemical Shift(1H)
- 15N - C- CO
H
R
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
