Molecular Biophysics

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Molecular Biophysics
Molecular structure by NMR (II)
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Nuclear Magnetic Resonance
RECAP
Some nuclei (e.g. 1H, 13C, 19F ) have certain
properties
Otto Stern
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RECAP
Import atoms in biomolecules
number symbol Electron configuration Electrons/shell Atomic weight Magnetic properties /Misc.
1 1H 2H (1p 1n) 1s1 1 1 75 of universe mass, spin ½ 2H spin 1
6 12C (6p6n) 13C (6p7n) 1s22s22p2 2,4 12 13C spin ½ (1)
7 14N (7p7n) 15N (7p8n) 1s22s22p3 2,5 14 14N spin 1 15N spin ½ (0.4)
8 16O (8p8n) 17O (8p9n) 1s22s22p4 2,6 16 17O spin 5/2 (0.04)
15 31P (15p16n) 1s22s22p6 3s23p3 2,8,5 31 spin ½
16 32S(16p16n) 33S(16p17n) 1s22s22p6 3s23p4 2,8,6 32 33S spin 3/2 (0.75)
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4
3
2
3
2
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Nuclear Magnetic Resonance
RECAP
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Nuclear Magnetic Resonance
RECAP
There is a torque due to the magnetic field
which keeps them precessing around the axis of
the applied magnetic field
t m x B
The precession frequency is proportional to the
magnetic field B w -g B Larmor frequency
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Nuclear Magnetic Resonance
RECAP
Difference in population
More nuclei align parallel with magnetic field
than antiparallel. This is called polarisation
E2
mI -1/2
N
E1
mI 1/2
Energy
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Nuclear Magnetic Resonance
RECAP
The difference results from the competition
between two different processes
A) The force of the external magnetic field which
makes the nuclei to align parallel to the
magnetic field.
B) The randomisation of the nuclei by thermal
fluctuations
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Nuclear Magnetic Resonance
RECAP
Torque on magnetic moment m generated by the
static magnetic field B
Potential energy of a magnetic moment m in a
static magnetic field B
Macroscopic system will fully align with
direction of B field 100 Polarisation
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Nuclear Magnetic Resonance
RECAP
In microscopic systems thermal fluctuations
pervent the magnetic moments to fully align.
Energy of thermal fluctuations kBT kB Bolzmann
constant 1.3810-23 m2 kg s-2 K-1
Therefore there is an almost equal chance for
parallel and antiparallel alignment (50
probability)
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Nuclear Magnetic Resonance
RECAP
Difference in population
More nuclei align parallel with magnetic field
than antiparallel.
E2
Sample magnetisation
Magnetic moments cancel but due to difference
small net magnetisation remains.
E1
Energy
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Nuclear Magnetic Resonance
RECAP
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Nuclear Magnetic Resonance
RECAP
The physical principle is identical to the
principle being used in a bicycle dynamo to
generate light in the bicycle lamp (Faradays
law)
w
change of magnetic flux due to precessing
magnetisation
voltage
Faradays law of induction
voltage
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Nuclear Magnetic Resonance
RECAP
The shape of the NMR signal
Voltage modulated by frequency w
Signal disappears with time (relaxation) There
are a number of time constants (T1, T2,
T2) describing various relaxation processes
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Nuclear Magnetic Resonance
RECAP
Analysis of signal
Freq. (MHz)
400
The frequency contained in the signal can be
obtained by mathematical analysis (Fourier
transformation)
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• Today
• Chemical shift
• Fine structure
• 2D experiments (brief)

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1H NMR spectrum of ethanol
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1H NMR spectrum of Lysozyme
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2D NMR spectroscopy
Cosy spectrum
Noesy spectrum
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Lysozyme NOESY spectrum
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15N 1H HSQC spectrum Lysozyme