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