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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 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
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
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