Protein Structure: NMR Spectroscopy

1
Protein Structure NMR Spectroscopy

• Microbiology 343
• David Wishart
• david.wishart_at_ualberta.ca

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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

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NMR Spectroscopy
Radio Wave Transceiver
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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)

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Electromagnetic Spectrum
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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

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NMR in Everyday Life
Magnetic Resonance Imaging
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NMR Spectroscopy
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Explaining NMR
UV/Vis spectroscopy
Sample
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Explaining NMR
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Each NMR Sample Contains 1023 Atoms with Protons
Inside
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Each Proton (and other nucleons) Has a Spin
Spin up Spin down
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Each Spinning Proton is Like a Mini-Magnet
Spin up Spin down
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Radio Waves Are Absrobed by Protons (Cause
Flipping)
N
N
hn
S
S
Low Energy High Energy
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The Spins Flip Back Forth With Different
Frequencies
Free Induction Decay
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The Bell Analogy
CH3
NH
CH
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Different Bells (Nuclei) Ring At Different
Frequencies
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What if You Ring All the Bells At Once?
FT
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How Do You Interpret All This Ringing? - FT NMR
Free Induction Decay
FT
NMR spectrum
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Fourier Transformation
iwt
F(w) f(t)e dt
Converts from units of time to units of frequency
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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

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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

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Bigger Magnets are Better
Increasing magnetic field strength
low frequency high frequency
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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
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NMR Magnet
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NMR Magnet Cross-Section
Sample Bore
Cryogens
Magnet Coil
Magnet Legs
Probe
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An NMR Probe
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NMR Sample Probe Coil
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1H NMR Spectra Exhibit...

• Chemical Shifts (peaks at different frequencies
  or ppm values)
• Splitting Patterns (from spin coupling)
• Different Peak Intensities ( 1H)

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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

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Characteristic Chemical Shifts
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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

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Spin-Spin Coupling
H
H
H
H
C - Y
C - CH
C - CH2
C - CH3
J
singlet doublet triplet
quartet
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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
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NMR Peak Intensities
Y
Y
Y

C - CH
C - CH2
C - CH3
AUC 1 AUC 2 AUC 3
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1H NMR Spectrum of a Small Molecule
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1H NMR Spectrum of a Large Molecule (Protein)
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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)

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2D Gels 2D NMR
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Multidimensional NMR
1D 2D 3D
MW 500 MW 10,000
MW 30,000
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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

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Multidimensional NMR
NOESY
TOCSY
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Assigning Chemical ShiftsTOCSY (white) NOESY
(red)
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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

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Measuring NOEs
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NMR Spectroscopy
Chemical Shift Assignments NOE
Intensities J-Couplings
Distance Geometry Simulated Annealing
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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

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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
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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
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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)

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Automated NMR Structure Generation
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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

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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