BSB 512

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BSB 512 Nuclear Magnetic Resonance
Spectroscopy TTh 220 - 325 pm Lecture notes
available at http//sos.bio.sunysb.edu/bsb512 Lec
ture 1 Basics Lecture 2-4 Protein
structure determination Lecture 5 Relaxation and
dynamics Lecture 6 Lab Session Sample
preparation. Pulse Programs Probe
selection. Tuning. Shimming Pulse
calibration. Data collection.
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References http//www.cis.rit.edu/htbooks/nmr Bo
oks Wuthrich, K. NMR of Proteins and Nucleic
Acids Levitt, MH Spin Dynamics Cavanagh J. et al.
Protein NMR Spectroscopy Ernst, R. et
al. Principles of NMR in One and Two
Dimensions Bax, A Two dimensional NMR in Liquids
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• NMR Spectroscopy Some history
• 1915 Einstein and de Hass - Correlation between
  magnetic moment and spin angular
  momentum
• 1922 Stern and Gerlach - Spins are quantized
• 1946 Bloch and Purcell - First NMR experiment
• Richard Ernst - Fourier transformations
• Jean Jeener - Two dimensional NMR - COSY
• 1976 Richard Ernst - First two dimensional NMR
  experiment
• 1986 Kurt Wuthrich - First independent NMR -
  X-ray comparison

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• High resolution solution NMR
• of proteins
• Observe protons (1H)
• This differs from x-ray
• diffraction where one
• determines structure based
• on the electron density from
• the electron rich atoms
• (C, N, O).
• Protein is solubilized in water.

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• High resolution solution NMR
• of proteins
• Observe protons
• Assign proton resonances to
• indivdual amino acids. Proton
• resonances are often resolved
• by differences in chemical
• shifts.
• Measure intra-residue and
• inter-residue proton to
• proton distances through
• dipolar couplings.
• Measure torsion angles
• through J-couplings.
• Use distance and torsion angle
• constraints to determine
• secondary and tertiary
• structure.

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• High resolution solution NMR
• of proteins
• Protons have a property called spin
• angular momentum.
• They behave like small bar magnets
• and align with or against a magnetic
• field.
• These small magnets interact with
• each other.

Bo
S
N
N
S
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13C and 15N also have spin angular momentum and
interact with 1H
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Magnetization can be transferred between 1H, 13C
and 15N to establish connectivities
H
H
H
C
Bo
C
N
C
H
H
Chemical Shifts J-couplings (through
bond) Dipolar couplings (through space)
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H
H
H
H
C
H
H
C
C
3D HSQC - NOESY for Inter-residue contacts
N
C
C
N
C
H
H
H
H
H
H
H
H
Magnetization can be transferred between 1H, 13C
and 15N to establish connectivities HNCA HNCOCA H
NCOCACB etc HSQC-TOCSY They all use INEPT
tranfers
C
H
H
C
C
N
C
C
N
C
H
H
H
H
Chemical Shifts J-couplings (through
bond) Dipolar couplings (through space)
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Concept 1 Some nuclei have non-zero spin
quantum numbers.
Nuclei with odd mass numbers have half-integer
spin quantum numbers. i.e. 13C, 1H, 31P are
spin I 1/2 17O is spin I
5/2 Nuclei with an even mass number and an even
charge number have spin quantum numbers of zero.
ie. 12C Nuclei with an even mass number and
an odd charge number have integer spin quantum
numbers. i.e. 2H is spin I 1 Electrons also
have a spin quantum number of 1/2
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Concept 2 Current passed through a coil induces
a magnetic field.
e-
e-
Concept 3 A changing magnetic field in a
coil induces a current.
e-
e-
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Concept 4 Placing nuclei with spin I 1/2 into
a magnetic field leads to a net magnetization
aligned along the magnetic field axis.
Mz
Classical picture
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Large external magnet
Net magnetization aligned along Z-axis of the
magnetic field
Bo
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The B1 field is produced by a small coil in the
NMR probe which is placed in the bore of the
large external magnet.
B
1
Bo
Net magnetization aligned along x-axis of the
magnetic field after application of B1 field.
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NMR magnet.
B
1
Bo
NMR probe
e-
e-
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Concept 5 When the B1 field is turned on, the
net magnetization rotates down into the XY plane
z
Bo
x
y
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Concept 6 When the B1 field is turned off, the
net magnetization relaxes back to the Z axis with
the time constant T1
T1 is the longitudinal relaxation time constant
which results from spin-lattice relaxation
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Exponential Functions
y
y e -x/t
x
y
y 1- e -x/t
x
Mz
Mz Mo (1- e -t/T1 )
t
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Concept 7 Individual spins precess about the
magnetic field axis.
z
Bo
x
y
Precession frequency Larmor frequency
wo -g Bo (MHz)
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Concept 8 After magnetization is rotated into
the xy plane by the B1 field produced from a
pulse through the coil, it will precess in the xy
plane.
y
z
Bo
x
x
y
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Concept 9 The individual magnetization vectors
whirling around in the xy plane represent a
changing magnetic field and will induce a current
in the sample coil which has its axis along the
x-axis.
y
y
x
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Concept 10 NMR signal is a Fourier transform of
the oscillating current induced in the sample
coil
x
y
y
-
y
-
x
time
frequency
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Chemical Shifts
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Concept 12 Nuclear spins produce small magnetic
fields
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Concept 13 Electrons are spin I 1/2 particles.
They produce small magnetic fields which oppose
the external magnetic field.
1H has a small chemical shift range (15 ppm).
113Cd has a large chemical shift range (300 ppm).
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What is a ppm? Ppm part per million
30 MHz
400 MHz
100 MHz
1H
13C
15N
1 ppm 400 Hz 15 ppm 6000 Hz
15 ppm
1H has a small chemical shift range (15 ppm).
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Concept 14 The surrounding electrons shield the
nuclear spins from the larger external Bo field.
This results in a reduction in the energy
spacing of the two energy levels and a lower
Larmor frequency. This is the chemical shift.
CH3
C-OH
b
a
frequency
CH3
C-OH
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Concept 11 In a frame of reference that ROTATES
at the Larmor (precession) frequency,
magnetization that is placed along the x-axis
does not move. (It simply relaxes back to the
z-axis via T1 processes.)
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b
b
100,010,000 Hz
100,000,000 Hz
a
a
CH3
C-OH
Reference or carrier 100,005,000 Hz
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Concept 15 The nuclei with different chemical
shifts and Larmor frequencies will rotate around
the z-axis at different speeds. T2 is the time
constant for the magnetization vectors to
"dephase" in the xy plane.
reference frequency
CH3
C-OH
frequency
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Chemical Shifts J-couplings (through
bond) Dipolar couplings (through space)
Structure
H
H
H
C
C
N
C
H
H
T1 relaxation T2 relaxation
Dynamics
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General One Dimensional Experiment
Acquire
t1
Fourier Transform t1 -gt f1
f1
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General One Dimensional Experiment
Acquire
t1
Fourier Transform t1 -gt f1
f1
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General One Dimensional Experiment
Acquire
t1
Fourier Transformation resolves multiple
frequencies that overlap in the time domain
Fourier Transform t1 -gt f1
f1
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General Two Dimensional Experiment
Acquire
t1
t2
Fourier Transform t1 -gt f1 and t2 -gt f2
f1
f2
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General Two Dimensional Experiment
Acquire
t1
t2
Vary t1 Collect a series of 1D spectra
Acquire
t1
t2
Acquire
t1
t2
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General Two Dimensional Experiment
t1
Vary t1 Collect a series of 1D spectra
f2
Here, the intensities of and do
not change as a function of the t1 evolution time
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General Two Dimensional Experiment
t1
Vary t1 Collect a series of 1D spectra
f2
Whereas here, the intensity of is modulated
as a function of the t1 evolution time
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General Two Dimensional Experiment
t1
Transpose and then Fourier transform in t1
dimension
f2
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General Two Dimensional Experiment
t1
Transpose and then Fourier transform in t1
dimension
f2
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General Two Dimensional Experiment
Projection on f2 gives original chemical shifts
f1
f2
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General Two Dimensional Experiment
Projection on f1 yields new information
f1
f2
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General Two Dimensional Experiment
J coupling
Dipolar coupling
1H chemical shift
1H chemical shift
13C chemical shift
1H chemical shift
1H chemical shift
1H chemical shift