Transcript BCHM300_NMR_1_2005 - Center for Structural Biology

01/24/05
Biomolecular Nuclear Magnetic
Resonance Spectroscopy
BASIC CONCEPTS OF NMR
• How does NMR work?
• Resonance assignment
• Structure determination
NMR text: 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, which arises from
the sub-atomic properties of neutrons and protons
• The nuclear spin angular momentum of each atom is
represented by a nuclear spin quantum number (I)
• All nuclei with odd mass numbers have I=1/2,3/2...
• Nuclei with even mass numbers and an even number of
protons have I=0
• Nuclei with even mass numbers and an odd number of
protons have I=1,2,3…
Biomolecular NMR: primarily spin 1/2 nuclei (1H, 13C, 15N, 31P)
Spin 1/2 Nuclei Aligned
in a Static Magnetic Field
Efficiency factornucleus
Ho
Energy
DE = h g Ho
Constants
Strength of
magnet
The Resonance Experiment
Ho
DE
H1
hn = DE
Equilibrium
Pump in energy
(RF transmitter)
Non-equilibrium
NMR signals
hn = DE
Release energy
(RF receiver)
Equilibrium
Magnetic Resonance Sensitivity
Sensitivity (S) ~ D(population)
S ~ DN =
Np
= e-DE/kT
Nap
DE is small
At room temp., DN ~ 1:105
Intrinsically low sensitivity
Need lots of sample
Efficiency factornucleus
DE = h g Ho
Constants
Strength of
magnet
Increase sensitivity by increasing magnetic field strength
Intrinsic Sensitivity of Nuclei
Nucleus
g
% Natural
Abundance
Relative
Sensitivity
1H
2.7 x 108
99.98
13C
6.7 x 107
1.11
0.004
15N
-2.7 x 107
0.36
0.0004
31P
1.1 x 108
100.
Prepare samples enriched in these nuclei
1.0
0.5
NMR Terminology
Chemical Shift & Linewidth
The exact resonance frequency (chemical shift) is
determined by the electronic environment of the nucleus
NMR Scalar and Dipolar Coupling
Through
Space
Through
Bonds
Coupling of nuclei gives information on structure
NMR Spectrum to 3D Structure
H
H
Interactions
Spectrum
H
H
H H H
H
H
H
H
Structure
Challenges For Determining
Protein Structures Using NMR
• Proteins have thousands of signals
• Assign the specific signal for each atom
• Thousands of interactions between atomsalso need to be assigned
• Need to transform from NMR spectrum
through interpretation of scalar and dipolar
interactions to generate 3D coordinates
Resonance Assignment
CH3-CH2-OH
OH
CH2 CH3
Which signal from which H atoms?
The key attribute: use the scalar and dipolar couplings to
match the set of signals with the molecular structure
Proteins Have Many Signals
1H
NMR Spectrum of Ubiquitin
~500 resonances
A large number of signals are overlapped
A Critical Feature of
Protein NMR Spectra
• Only some nuclei are coupled
Each amino acid gives rise to an independent NMR
sub-spectrum, which is much simpler than the
complete protein spectrum
Methods have been
developed to extract
each sub-spectrum
from the whole
Basic Strategy to Assign
Resonances in a Protein
1. Identify resonances for each amino
acid
T G L S
R G
S
2. Put amino acids in order
- Sequential assignment (R-G-S,T-L-G-S)
- Sequence-specific assignment
1
2
3
4
5
6
7
R-G-S-T-L-G-S
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
are Further Dispersed by the 3D Fold
What would the unfolded protein look like?
Proteins Have Overlapped Signals
1H
NMR Spectrum of Ubiquitin
Resolve resonances by multi-dimensional experiments
Resolve Peaks By Multi-D NMR
A BONUSregions in
2D spectra provide
protein fingerprints
If 2D cross peaks
overlap go to 3D
or 4D …..
Solution to the Protein Challenge
1. Increase dimensionality of spectra to
better resolve signals: 1234
2. Detect signals from heteronuclei
(13C,15N)
t2
t1
t3
Heteronuclear nD NMR
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
Structure Determination by NMR
NMR Experimental Observables
Providing Structural Information
• Backbone conformation from chemical
shifts (Chemical Shift Index- CSI): ,
• Distance restraints from NOEs
• Hydrogen bond restraints
• Backbone and side chain dihedral angle
restraints from scalar couplings
• Orientation restraints from residual
dipolar couplings
NMR Structure Calculations
• Objective is to determine all conformations
consistent with the experimental data
• Programs that only do conformational search
lead to bad chemistry  use molecular force
fields improve molecular properties
 Some programs try to do both at once
 Need a reasonable starting structure
• NMR data is not perfect: noise, incomplete
data  multiple solutions (conformational
ensemble)
Variable Resolution of Structures
• Secondary structures well defined, loops variable
• Interiors well defined, surfaces more variable
• Trends the same for backbone and side chains
Restraints and Uncertainty
Large # of restraints
= low values of RMSD
Large # of restraints
for key hydrophobic
side chains
Assessing the Quality
of NMR Structures
• Number of experimental constraints
• RMSD of structural ensemble (subjective!)
• Violation of constraints- number, magnitude
• Molecular energies
• Comparison to known structures: PROCHECK
• Back-calculation of experimental parameters