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Protein Structure: NMR Spectroscopy

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Title: Protein Structure: NMR Spectroscopy


1
Protein Structure NMR Spectroscopy
  • Microbiology 343
  • David Wishart
  • david.wishart_at_ualberta.ca

2
Objectives
  • To learn about the basic principles of NMR
    spectroscopy To gain an awareness of what an NMR
    spectrum looks like and why
  • To gain a basic understanding of how NMR can be
    used to determine protein structures, along with
    its strengths/weaknesses

3
NMR Spectroscopy
Radio Wave Transceiver
4
Principles of NMR
  • Measures nuclear magnetism or changes in nuclear
    magnetism in a molecule
  • NMR spectroscopy measures the absorption of light
    (radio waves) due to changes in nuclear spin
    orientation
  • NMR only occurs when a sample is in a strong
    magnetic field
  • Different nuclei absorb at different energies
    (frequencies)

5
Electromagnetic Spectrum
6
Different Types of NMR
  • Electron Spin Resonance (ESR)
  • 1-10 GHz (frequency) used in analyzing free
    radicals (unpaired electrons)
  • Magnetic Resonance Imaging (MRI)
  • 50-300 MHz (frequency) for diagnostic imaging of
    soft tissues (water detection)
  • NMR Spectroscopy (MRS)
  • 300-900 MHz (frequency) primarily used for
    compound ID and characterization

7
NMR in Everyday Life
Magnetic Resonance Imaging
8
NMR Spectroscopy
9
Explaining NMR
UV/Vis spectroscopy
Sample
10
Explaining NMR
11
Each NMR Sample Contains 1023 Atoms with Protons
Inside
12
Each Proton (and other nucleons) Has a Spin
Spin up Spin down
13
Each Spinning Proton is Like a Mini-Magnet
Spin up Spin down
14
Radio Waves Are Absrobed by Protons (Cause
Flipping)
N
N
hn
S
S
Low Energy High Energy
15
The Spins Flip Back Forth With Different
Frequencies
Free Induction Decay
16
The Bell Analogy
CH3
NH
CH
17
Different Bells (Nuclei) Ring At Different
Frequencies
18
What if You Ring All the Bells At Once?
FT
19
How Do You Interpret All This Ringing? - FT NMR
Free Induction Decay
FT
NMR spectrum
20
Fourier Transformation
iwt
F(w) f(t)e dt
Converts from units of time to units of frequency
21
Which Elements or Molecules are NMR Active?
  • Any atom or element with an odd number of
    neutrons and/or an odd number of protons
  • Any molecule with NMR active atoms
  • 1H - 1 proton, no neutrons, AW 1
  • 13C - 6 protons, 7 neutrons, AW 13
  • 15N - 7 protons, 8 neutrons, AW 15
  • 19F 9 protons, 10 neutrons, AW 19

22
The NMR Equation
n gB/2p
  • B magnetic field strength in Tesla (1 Tesla
    10,000 Gauss 1000 kitchen magnets)
  • g gyromagnetic ratio (characteristic of each
    nucleus, each atom in a molecule

23
Bigger Magnets are Better
Increasing magnetic field strength
low frequency high frequency
24
Different Isotopes Absorb at Different
Frequencies
2H
15N
13C
19F
1H
30 MHz 50 MHz 125 MHz 480 MHz
500 MHz
low frequency high frequency
25
NMR Magnet
26
NMR Magnet Cross-Section
Sample Bore
Cryogens
Magnet Coil
Magnet Legs
Probe
27
An NMR Probe
28
NMR Sample Probe Coil
29
1H NMR Spectra Exhibit...
  • Chemical Shifts (peaks at different frequencies
    or ppm values)
  • Splitting Patterns (from spin coupling)
  • Different Peak Intensities ( 1H)

30
Chemical Shifts
  • Key to the utility of NMR in chemistry
  • Different 1H in different molecules exhibit
    different absorption frequencies
  • Arise from the electron cloud effects of nearby
    atoms or bonds, which act as little magnets to
    shift absorption n up or down
  • Mostly affected by electronegativity of
    neighbouring atoms or groups

31
Characteristic Chemical Shifts
32
Spin-Spin Coupling
  • Many 1H NMR spectra exhibit peak splitting
    (doublets, triplets, quartets)
  • This splitting arises from adjacent hydrogens
    (protons) which cause the absorption frequencies
    of the observed 1H to jump to different levels
  • These energy jumps are quantized and the number
    of levels or splittings n 1 where n is the
    number of nearby 1Hs

33
Spin-Spin Coupling
H
H
H
H
C - Y
C - CH
C - CH2
C - CH3
J
singlet doublet triplet
quartet
34
Spin Coupling Intensities
1 1 1 1 2 1 1 3 3 1 1 4 6 4 1 1 5 10 10 5 1
3 3
2
1 1
1 1
1 1
Pascals Triangle
35
NMR Peak Intensities
Y
Y
Y

C - CH
C - CH2
C - CH3
AUC 1 AUC 2 AUC 3
36
1H NMR Spectrum of a Small Molecule
37
1H NMR Spectrum of a Large Molecule (Protein)
38
NMR of Big Molecules
  • Too many peaks overlapping one another to easily
    interpret
  • Difficult to extract peak intensity information
  • Peaks are broad and generally lose all the fine
    details (J-coupling)
  • Is there a way of spreading out all these peaks?
    (Yes! 2D NMR)

39
2D Gels 2D NMR
40
Multidimensional NMR
1D 2D 3D
MW 500 MW 10,000
MW 30,000
41
The NMR Process
  • Obtain protein sequence
  • Collect TOCSY NOESY data
  • Use chemical shift tables and known sequence to
    assign TOCSY spectrum
  • Use TOCSY to assign NOESY spectrum
  • Obtain inter and intra-residue distance
    information from NOESY data
  • Feed data to computer to solve structure

42
Multidimensional NMR
NOESY
TOCSY
43
Assigning Chemical ShiftsTOCSY (white) NOESY
(red)
44
The NOE
  • NOEs build up over time (50-400 ms)
  • NOEs are stronger for 1H atoms that are closer
    together in space
  • NOEs are weaker for 1H atoms far away from each
    other
  • Limit is from 1.5-5.5 Angstroms

45
Measuring NOEs
46
NMR Spectroscopy
Chemical Shift Assignments NOE
Intensities J-Couplings
Distance Geometry Simulated Annealing
47
NMR Spectroscopy Protein Structure
  • NMR generates multiple structures (called an
    ensemble) instead of just one structures (as in
    X-ray)
  • This reflects the fact that there are multiple
    solutions to the set of distance (NOE,
    J-coupling, Hbond) restraints provided to the
    computer
  • The more blurred the region or loop, the more
    flexible it likely is

48
The Final Result
ORIGX2 0.000000
1.000000 0.000000 0.00000
2TRX 147 ORIGX3
0.000000 0.000000 1.000000 0.00000
2TRX 148 SCALE1
0.011173 0.000000 0.004858 0.00000
2TRX 149 SCALE2
0.000000 0.019585 0.000000 0.00000
2TRX 150
SCALE3 0.000000 0.000000 0.018039
0.00000 2TRX 151
ATOM 1 N SER A 1 21.389
25.406 -4.628 1.00 23.22 2TRX 152
ATOM 2 CA SER A 1
21.628 26.691 -3.983 1.00 24.42 2TRX 153
ATOM 3 C SER A 1
20.937 26.944 -2.679 1.00 24.21 2TRX
154 ATOM 4 O SER A
1 21.072 28.079 -2.093 1.00 24.97
2TRX 155 ATOM 5 CB
SER A 1 21.117 27.770 -5.002 1.00 28.27
2TRX 156 ATOM 6
OG SER A 1 22.276 27.925 -5.861 1.00
32.61 2TRX 157 ATOM
7 N ASP A 2 20.173 26.028 -2.163
1.00 21.39 2TRX 158
ATOM 8 CA ASP A 2 19.395 26.125
-0.949 1.00 21.57 2TRX 159
ATOM 9 C ASP A 2 20.264
26.214 0.297 1.00 20.89 2TRX 160
ATOM 10 O ASP A 2
19.760 26.575 1.371 1.00 21.49 2TRX 161
ATOM 11 CB ASP A 2
18.439 24.914 -0.856 1.00 22.14 2TRX 162
49
How Long Does it Take?
Chemical Shift Assignments NOE
Intensities J-Couplings
Distance Geometry MD Refinement
Evaluation
6-12 months 2-4 months 1-2 months
50
High Throughput NMR
  • Higher magnetic fields (From 400 MHz to 900 MHz)
  • Higher dimensionality (From 2D to 3D to 4D)
  • New pulse sequences (TROSY, CBCA(CO)NH)
  • Improved sensitivity
  • New parameters (Dipolar coupling, cross
    relaxation)

51
Automated NMR Structure Generation
52
X-ray Versus NMR (Bottlenecks)
X-ray NMR
  • Producing enough protein for trials
  • Crystallization time and effort
  • Crystal quality, stability and size control
  • Finding isomorphous derivatives
  • Chain tracing checking
  • Producing enough labeled protein for collection
  • Sample conditioning
  • Size of protein
  • Assignment process is slow and error prone
  • Measuring NOEs is slow and error prone

53
Conclusions
  • Approximately ¼ of all structure in the PDB have
    been generated by NMR
  • Proteins as large as 700 residues have been
    analyzed/solved by NMR
  • Excellent method for studying protein structure
    in solution, probing dynamics, flexibility,
    stability, kinetics and rate processes
  • NMR compliments X-ray work
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