Nuclear Magnetic Resonance Spectroscopy

1
Nuclear Magnetic Resonance Spectroscopy

• Prof. Chad M. Rienstra
• With special thanks to
• Kathryn D. Kloepper

2
Outline

• Introduction to NMR
• Solution vs. Solid-state
• Research Examples
• Parkinsons Disease
• Alpha-Synuclein

3
Objectives

• What process is observed in an NMR signal?
• Describe the process of resonance
• How does Boltzmann relate to NMR?
• How or why do we go from the time to frequency
  domain?
• What is chemical shift and how is it useful?
• When are different chemical shifts observed?
• Why define chemical shift relative to a reference
  compound?
• How do coupling networks define structure of
  molecules?
• Scalar couplings transmit energy through bonds
• Dipolar couplings transmit energy through space

4
Nuclear Magnetic Resonance

• Multidisciplinary spectroscopic technique
• Takes advantage of properties of the nucleus in
  order to get chemical information
• Uses very high field magnets and
  computer-controlled equipment

750 MHz NMR spectrometer at U Illinois
5
NMR v. MRI

• Used to be called nuclear MRI

http//www.medinnovations.usa.siemens.com/procedur
es/mri/
6
Slight Public Relations Problem

• Used to be called nuclear MRI

7

• N
• M
• R

8

• Nuclear
• Magnetic
• Resonance

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Nuclear spin

• Electrons, protons, and neutrons have spin
• Overall spin (I) of nucleus determined by numbers
  of neutrons and protons
• Examples of spin 1/2 nuclei 1H, 13C,15N, 19F,
  31P

1. Number of of neutrons and number of protons
are even, then nucleus has spin of 0. 2. Number
of neutrons plus number of protons are odd, then
nucleus has spin of half integer (1/2, etc). 3.
Number of neutrons and number of protons are odd,
then nucleus has spin of whole integer (1, 2,
etc).
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Most Elements Have Nuclear Spin
http//www.mrl.ucsb.edu/mrl/centralfacilities/spec
troscopy/nmr_table_mrl.html
11
Splitting of Energy Levels

• Nucleus of spin I has 2I1 possible orientations
• Spin 1/2 has 2 possible orientations
• When magnetic field is applied, the energy levels
  split
• orient with (?) or against (?) the magnetic field

Applied magnetic field
No field
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Splitting of Energy Levels

• Nucleus of spin I has 2I1 possible orientations
• Spin 1/2 has 2 possible orientations
• When magnetic field is applied, the energy levels
  split
• orient with (?) or against (?) the magnetic field
• Which orientation will be lower energy?

Applied magnetic field
No field
13
Splitting of Energy Levels

• Nucleus of spin I has 2I1 possible orientations
• Spin 1/2 has 2 possible orientations
• When magnetic field is applied, the energy levels
  split
• orient with (?) or against (?) the magnetic field
• Which orientation will be lower energy?
• Aligning with (?) the magnetic field

Applied magnetic field
No field
?E
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Populations of energy levels

• The Boltzmann distribution describes the relative
  populations of energy levels at a given
  temperature

Ratio of populations
Applied magnetic field
No field
?E
15
Populations of energy levels

• The Boltzmann distribution describes the relative
  populations of energy levels at a given
  temperature

Ratio of populations
Applied magnetic field
No field
?E
16
Populations of energy levels

• The Boltzmann distribution describes the relative
  populations of energy levels at a given
  temperature

Ratio of populations
Simplify expression
Applied magnetic field
No field
?E
17
Populations of energy levels

• The Boltzmann distribution describes the relative
  populations of energy levels at a given
  temperature

Ratio of populations
Simplify expression
Plug in for ?
Applied magnetic field
No field
?E
18
Populations of energy levels

• The Boltzmann distribution describes the relative
  populations of energy levels at a given
  temperature

Ratio of populations
Simplify expression
Plug in for ?
Applied magnetic field
No field
?E
19
Populations of energy levels

• Calculate relative populations of energy levels
  at room temperature. T 300 K B0 21 T
  ? 27x107 radT-1s-1

Ratio of populations
Applied magnetic field
No field
?E
20
Populations of energy levels

• Calculate relative populations of energy levels
  at room temperature. T 300 K B0 21 T
  ? 27x107 radT-1s-1

Ratio of populations
Find ?E
Applied magnetic field
No field
?E
21
Populations of energy levels

• Calculate relative populations of energy levels
  at room temperature. T 300 K B0 21 T
  ? 27x107 radT-1s-1

Ratio of populations
Find ?E
Applied magnetic field
No field
?E
22
Populations of energy levels

• Calculate relative populations of energy levels
  at room temperature. T 300 K B0 21 T
  ? 27x107 radT-1s-1

Ratio of populations
Find ?E
6.0 x 10-25 J
0.36 J / mol
Applied magnetic field
No field
?E
23
Populations of energy levels

• Calculate relative populations of energy levels
  at room temperature. T 300 K B0 21 T
  ? 27x107 radT-1s-1

Ratio of populations
Find ?E
6.0 x 10-25 J
0.99986
Plug in ?E and solve
Applied magnetic field
No field
?E
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NMR With Any Magnetic Field
http//www.earthsfield-nmr.com

• Earths magnetic field
• 0.3 Gauss near equator
• 0.6 Gauss near poles
• Practical uses
• Distinguishing oilfrom water
• Imaging shapesunderground
• Superconducting magnets
• Wire alloys
• Niobium titanium
• Niobium tin
• Liquid helium cryostat
• Vacuum jacket
• 4.2 Kelvin
• 21 Tesla 210,000 Gauss

http//nmr.chem.umn.edu/cutaway.html
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900 MHz Magnet in Richland, WA

• http//www.pnl.gov/news/2002/nmr.htm

16 tons, 10 years to manufacture Several miles of
wire () 10,000,000
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Nuclear Magnetic Resonance

• Apply radiowaves at same frequency as difference
  in energy levels
• Causes some spins to go to higher energy state
• Called a spin flip
• Turn off radiowaves, spins will return to
  equilibrium
• Relax back to lower energy level

time
RF pulse
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• To what part of the electromagnetic radiation
  spectrum does this correspond?
• ?E 6.0 x 10-25 J h?
• ? ?E / h (6.0 x 10-25 J / 6.626 x 10-34 Js)
• ? 9.0 x 108 Hz 900 MHz

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Free induction decay

• This induces a voltage in the detection coil
  around the sample free induction decay (FID)

time
Figure www.chem.umn.edu/class/5361/que04f/NMR_bas
ics1.pdf
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Fourier Analysis of Piano (A3, 220 Hz)

• Prominent frequency of 220 Hz
• Overtones depend upon acoustics of piano
• Tuning depends on the length of wire and tension

http//static.flickr.com/95/249272111_57ec4e8440_b
.jpg
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Fourier transform NMR spectroscopy

• Can write the FID as

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Fourier transform NMR spectroscopy

• Can write the FID as
• To get the NMR spectrum, perform a Fourier
  transform

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Fourier transform NMR spectroscopy

• Every frequency component in the FID yields a
  line in the absorption spectrum at the precession
  frequency

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Fourier transform NMR spectroscopy

• Every frequency component in the FID yields a
  line in the absorption spectrum at the precession
  frequency

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Fourier transform NMR spectroscopy

• Every frequency component in the FID yields a
  line in the absorption spectrum at the precession
  frequency

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Fourier transform NMR spectroscopy

• Every frequency component in the FID yields a
  line in the absorption spectrum at the precession
  frequency

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Fourier transform NMR spectroscopy

• Every frequency component in the FID yields a
  line in the absorption spectrum at the precession
  frequency

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Fourier transform NMR spectroscopy

• Every frequency component in the FID yields a
  line in the absorption spectrum at the precession
  frequency

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Chemical shift

• Electron density around nucleus reduces resonance
  frequency of the nuclear spin shielding
• Frequency given by
• ? depends on electron density around nucleus
  changes depending on neighbors
• Leads to shift in frequency
• Chemical shifts are dependent on chemical
  environment

where ? shielding constant
acetone
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Chemical shift

• More convenient to characterize the frequency
  shift relative to a reference compound
• Therefore, ? is independent of frequency

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Chemical shift

• More convenient to characterize the frequency
  shift relative to a reference compound
• Therefore, ? is independent of frequency
• Why is that important?

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Chemical shift

• More convenient to characterize the frequency
  shift relative to a reference compound
• Therefore, ? is independent of frequency
• Why is that important?

Because then all measurements using different
spectrometers with different magnetic fields will
result in the same ?!
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Chemical shifts depend on environment
acetone
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Chemical shifts depend on environment
acetone
benzene
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Structure of Ethane
Example Ethane Step 1 Draw the molecular
structure
Step 2 Count number of different protons One!
Proton spectrum of CH3CH3
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Structure of Ethanol
Example Ethanol Step 1 Draw the molecular
structure
Step 2 Count number of different protons Three!
1. CH3 2. CH2 3. OH Proton spectrum of
CH3CH2OH
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Shielding Depends Upon Structure
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Objectives

• What process is observed in an NMR signal?
• Describe the process of resonance
• Nuclear spins are excited by radiowaves into a
  higher energy state and transition back to the
  lower energy state (relaxation)
• How does Boltzmann relate to NMR?
• The Boltzmann distribution gives the relative
  populations of low and high energy levels
• How or why do we go from the time to frequency
  domain?
• Fourier Transforming the free induction decay
  spectrum yields frequencies. The frequency
  domain is easier to analyze.
• What is chemical shift and how is it useful?
• When are different chemical shifts observed?
• When there are different chemical environments.
• Why define chemical shift relative to a reference
  compound?
• So chemical shifts from different spectrometers
  and magnetic field strengths can be compared.

48
Nuclear Magnetic Resonance Using the Power of
Magnets to Investigate Protein Structure

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Outline

• Introduction to NMR
• Solution vs. Solid-state
• Research Examples
• Parkinsons Disease
• Alpha-Synuclein

50
Protein structures by NMR

• NMR can be used to determine three dimensional
  structures of proteins
• Determine distribution of structural features and
  how they are connected
• IMPORTANT Knowing a proteins structure is key
  to understanding its function!

1o
2o
3o
Image from www.genome.gov
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Solution NMR General Protocol

• 1. Isotopically label protein (15N, 13C)
• 2. Assign the chemical shifts
• 3. Collect restraints
• 4. Calculate structure
• 5. Refine (repeat)

This has been done over 5,000 times in
solution! (http//www.pdb.org/pdb/statistics/holdi
ngs.do)
Kumar, A. Ernst, R.R. Wüthrich, K. Biochem.
Biophys. Res. Comm. 1980, 95, 16. Williamson,
M.P. Havel, T.F. Wüthrich, K., J. Mol. Biol.
1985, 182, 295315.
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Mapping Structures of Proteins
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Solid-state NMR in Structural Biology
Many proteins cannot be studied by the
traditional structural methods (X-ray
crystallography or solution NMR)
Nano/microcrystalline globular proteins
Membrane proteins
Fibrils
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Solution vs. Solid-state NMR

• Solid-state NMR
• samples go up bottom
• no size limit on samples
• broader linewidths

• Solution NMR
• samples dropped from top
• size limitation
• very narrow linewidths

Solution NMR sample tube
Solid-state NMR probe
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Magic-Angle Spinning Solid-State NMR

• Solid-state NMR (SSNMR) yields very broad spectra
  
• If sample is spun very fast at the magic angle
  (54.7o) then can collapse the powder pattern into
  sharp, narrow peaks

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Synuclein

• Synuclein a 14 kDa (140 amino acids) protein
  found in your brain
• Function not known but something causes synuclein
  to aggregate in diseased brains
• Linked to several neurodegenerative diseases

Location of synuclein in neuron
Image by K. Kloepper
57
Synuclein and Parkinsons Disease

• Synuclein aggregates involved in Parkinsons
  diseasebut how exactly?
• Mutations in protein cause early-onset
  Parkinsons
• Synuclein main component of Lewy bodies
• Need structural information to determine role in
  disease!

4 um
Synuclein aggregates
Lewy body
From Kloepper, et al., Protein Expres. Purif.
2006, 48, 112-117.
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Stable Isotope (13C, 15N) Incorporation

• 13C and 15N required to perform NMR
• Natural abundance of isotopes too low
• 13C 1.11, 15N 0.37
• Use E. coli to express large quantities of
  labeled synuclein
• Select for cells that incorporate the plasmids by
  growing on antibiotic-containing agar plates
• This transformation takes about 2 h
• of lab steps and an overnight incubation

Kloepper, et al. Protein Expr. Purif. 2006,
48(1), 112-117.
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Stable Isotope (13C, 15N) Incorporation

• Transfer a colony to growth media and let
  bacteria replicate
• Initial growth in rich media (LB) containing lots
  of nutrients
• Transfer to minimal media
• 13C-glucose
• 15N-ammonium chloride
• !
• Induce for a few hours-- tells the bacteria
• to start making synuclein
• Growth takes 12 h from inoculation with colony
  to spinning down cells

Kloepper, et al. Protein Expr. Purif. 2006,
48(1), 112-117.
60
Purification of Synuclein

• After lysing (breaking open) the cells, heat
  purify and precipitate synuclein by overnight
  salting out
• Two chromatography steps hydrophobic interaction
  and size exchange (sizing)

Kloepper, et al. Protein Expr. Purif. 2006,
48(1), 112-117.
61
Preparation of Fibril Samples

• Fibrils prepared in vitro by incubating at 37 oC
  with shaking
• Ultracentrifuge to obtain fibril pellet and
  transfer into NMR rotor

fibril pellet
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SSNMR of Fibril Samples

• Better experiments than one dimensional 13C
  spectra required

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Two-Dimensional SSNMR
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Identification of Amino Acids
Chemical shifts depend on chemical environment
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Identification of Amino Acids
Chemical shifts depend on chemical environment
Alanines
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Identification of Amino Acids
Chemical shifts depend on chemical environment
Threonines
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Need More Resolution!
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Backbone Walk
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Structural Model and Refinement

• Use chemical shift assignments to determine
  distances between amino acids
• Use distances to get initial structure
• Refine the structure through an iterative process

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Acknowledgments

• (Dr.) Kathryn D. Kloepper
• National Institutes of Health ()
• Undergraduate researchers
• Kem Winter, Kevin Hartman, Daniel Ladror, Reika
  Ebisu

Kem Winter June 04 - Aug 05 Wisconsin Ph.D.
Analytical
Kevin Hartman Jan 05 - Aug 07 Berkeley Ph.D.
Physical
Daniel Ladror Jan 05 - present ???? Ph.D.
Physical
Reika Ebisu Jan 07 - present Minnesota Pharmacy
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Amino acids
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2D pulse sequence
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