Nuclear Magnetic Resonance (NMR) Spectroscopy Kurt Wuthrich Chemistry-2002 Richard Ernst Chemistry -1991 Felix Bloch & Edward Purcell Physics-1952 Paul Lauterbur & Peter Mansfield Medicine-2003 Brian Sykes Lewis Kay
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Nuclear Magnetic Resonance (NMR) Spectroscopy
Kurt WuthrichChemistry-2002
Richard ErnstChemistry -1991
Felix Bloch & Edward PurcellPhysics-1952
Paul Lauterbur & Peter MansfieldMedicine-2003
Brian Sykes Lewis Kay
Principles of NMR Protein Spectroscopy
What does NMR tell us ?
1) Primary structure characterization2) Dynamics - psec-sec timescales3) Equilibrium binding4) Folding/Unfolding5) Three dimensional structure
Some Advantages 1) Solution based2) Non-destructive3) Residue specific information
Principles of NMR Protein Spectroscopy
Wavelength (nm)
10 100 1000 104 105 106 107 108 109
UV/Vis
IR NMR
Nuclear spintransitions
Electrontransitions
Crystallography
X-rays Radio waves
Principles of NMR Protein Spectroscopy
Absorption of energy by nucleus - depends on nuclear spin (I) = sum of unpaired protons + neutrons (spin 1/2)
1H 1/2 1 012C 0 6 613C 1/2 6 714N 1 7 715N 1/2 7 8
Nucleus I # Protons # Neutrons
I≠0 - NMR observed - spin will have magnetic moment µ=γI For proteins 1H, 13C, 15N (31P)
Principles of NMR Protein Spectroscopy
mI = -1/2 β
N
S
S
NN
S
m=(-I, -I+1 ….I-1, I)
mI = +1/2 α
Bo
Principles of NMR Protein Spectroscopy
x
y
z
Bo
µ=-γIz
Normal Magnetic Field (Bo)
Principles of Protein NMR Spectroscopy
mI = -1/2 β
N
S
S
NN
SmI = +1/2 α
So how does this give us an “NMR signal” ?
α
β
Principles of Protein NMR Spectroscopy
α
β
This occurs for all 1H, 13C, 15N in magnetic field
B0
1H 26.75 13C 6.73
15N -2.72
γ (∗107 rad / T sec)
Principles of Protein NMR Spectroscopy
α
β
ΔE = γhB0 = hν ν = γB02π
(Hz) ω = γB0 (rad)
Larmor Precession
1H 26.75 600.00 13C 6.73 150.8715N -2.71 60.82
γ (∗107 rad / T sec)Nucleus ν at 14.09 T
Principles of Protein NMR Spectroscopy
α
β
NMR is an insensitive method 1H N=106
11.75T 18.79T499,980
500,020
499,968
500,032
Principles of Protein NMR Spectroscopy
Sensitivity
nα-nβ = Δn = NγhBo
2kTMo=nαµzα + nβµzβ = Δnµzα= 1 γhΔn = 1 N (γh)2Bo
2 4kT
x
y
z
Bo
x
y
z
Bo
Principles of Protein NMR Spectroscopy
Net Magnetization
x
y
z
x
y
z
Mo
Bo Boωo
Rotating Frame
Principles of Protein NMR Spectroscopy
How is the NMR Signal Obtained ?
x
y
z
Mo
Bo
-employ a rf pulse at ν of desired nucleus
x
y
z
“rf pulse”
θ
B1 B1
Principles of Protein NMR Spectroscopy
x
y
z
Mo
Box
y
z
θ = π/2π/2
rfon off
B1 B1
pw = 6 µsec γB1 = 41 kHz
Principles of Protein NMR Spectroscopy
x
y
z
π/2
rfon off
B1
x
y
z
Mo
Bo
B1
x
y
z
B1receiver
FT
time ν
Principles of Protein NMR Spectroscopy
x
y
z
off
B1
x
y
z
B1
receiver
time
T1
x
y
z
B1
T2
Mz(t) = Mo(1-e-t/T1)
My(t) = My(0)*e-t/T2
Principles of Protein NMR Spectroscopy
100 10 1 0.1 0.01 0.001Correlation Time, τc (nsec)
Molecular Weight
T1, T2 (sec)
100
10
1
0.1
0.01
0.001
T1
T2
Linewidths
MW 100
0.5 Hz
MW 20,000
10 Hz
Principles of Protein NMR Spectroscopy
NMR Instrumentation
Magnet - Bo 18.79 T
vacuum
N2(l)
He(l)
magnet
22.31 T (2006)probe
Principles of Protein NMR Spectroscopy
Magnet Technology
Principles of Protein NMR Spectroscopy
Magnet Technology
Principles of Protein NMR Spectroscopy
Probe Technology
Bo
B1
Principles of Protein NMR Spectroscopy
Cold “Cryogenic” Probe
S/N ~ 1/{Rs*(Ts+Tpa)+(Rc*(Tc +Tpa)}1/2
Tpa, Tc - lowered 298˚K 20˚K
Rs, Ts - near 298˚K
3-4 time more sensitive
500 MHz + cold probe = 1.6x S/N 800 MHz
Principles of Protein NMR Spectroscopy
Block Diagram of NMR Spectrometer
Probe
Transmitter Preamplifier
Duplexer
CPU Receiver
Computer
obs, lk
obs, lkobs, dec, lk
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