Nuclear Magnetic Resonance (NMR) Spectroscopy Kurt Wuthrich Chemistry-2002 Richard Ernst Chemistry...

Nuclear Magnetic Resonance (NMR) Spectroscopy

Kurt WuthrichChemistry-2002

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Richard ErnstChemistry -1991

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Felix Bloch & Edward PurcellPhysics-1952

Paul Lauterbur & Peter Mansfield

Medicine-2003

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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


Wavelength (nm)

10 100 1000 104 105 106 107 108 109

UV/Vis

IR NMR

Nuclear spin

transitions

Electron

transitions

Crystallography

X-rays

Radio waves


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 6

13C 1/2 6 714N 1 7 715N 1/2 7 8

Nucleus I # Protons # Neutrons

I≠0 - NMR observed - spin will have magnetic moment =I For proteins 1H, 13C, 15N (31P)


I = 1/2= Iz

= mIh

mI = -1/2

Under normal conditions the difference between these is negligible

N

S

S

N

Now lets put these in a static magnetic field

N

S

m=(-I, -I+1 ….I-1, I)

mI = +1/2

Bo


x

y

z

Bo

In a magnetic field E = -Bo

= -IzBo = -hmIBo m=(-I, -I+1 ….I-1, I)

=-Iz

Normal Magnetic Field (Bo)

Principles of Protein NMR Spectroscopy

mI = -1/2

N

S

S

NN

SmI = +1/2

So how does this give us an “NMR signal” ?

E = 1hB0

2 -hmIB0- 1hB0

2

E = hB0


E = hB0

This occurs for all 1H, 13C, 15N in magnetic field

B0 = 11.75 T (500 MHz) 14.09 T (600 MHz) 18.79 T (800 MHz)

B0

1H 26.75 13C 6.73

15N -2.72

rad / T sec)

As B0 so does E !


E = hB0 = h

We can use this to select for the nucleus of interest

= B0

2(Hz)

= B0(rad)

Larmor Precession

Use frequency to stimulate transitions !

1H 26.75 600.00 13C 6.73 150.87

15N -2.71 60.82

rad / T sec)

Nucleus at 14.09 T


E = hB0 = h

NMR is an insensitive method

Populations of determined by E - Boltzman distribution

n-n = n = NhB0

2kT

1H N=106

11.75T 18.79T

499,980

500,020

499,968

500,032

n = 40 n = 64

Only measure net difference (n)InsensitivityLarger magnet = sensitivity


Sensitivity

n-n = n = NhBo

2kTMo=nz + nz = nz= 1 hn = 1 N (h)2Bo

2 4kT

- it turns out the observed signal (S) - varies with Bo2

x

y

z

Bo

x

y

z

Bo


Net Magnetization

x

y

z

x

y

z

Mo

Bo Boo

Rotating Frame


How is the NMR Signal Obtained ?

x

y

z

Mo

Bo

-employ a rf pulse at of desired nucleus

x

y

z

2*pw*B1=

“rf pulse”

B1 B1


x

y

z

Mo

Box

y

z

2*pw*B1=

= /2

rfon off

pw= 1 4*B1

B1 B1

pw = 6 sec B1 = 41 kHz


x

y

z

rfon off

B1

x

y

z

Mo

Bo

B1

x

y

z

B1receiver

FT

time


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


100 10 1 0.1 0.01 0.001Correlation Time, c (nsec)

Molecular Weight

T1, T2 (sec)

100

10

1

0.1

0.01

0.001

T1

T2

1/2 = 1 T2*

Linewidths

MW 100

0.5 Hz

MW 20,000

10 Hz


NMR Instrumentation

Magnet - Bo 18.79 T

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vacuum

N2(l)

He(l

)magnet

22.31 T (2006)probe


Magnet Technology

Ribonuclease (1957) - 40 MHzLysozyme (1995) - 750 MHz

0.94 T 117.62 T 35122.31 T 563

Field S/N


Probe Technology







Bo

B1


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


Block Diagram of NMR Spectrometer

Probe

Transmitter Preamplifier

Duplexer

CPU Receiver

Computer

obs, lk

obs, lkobs, dec, lk

Nuclear Magnetic Resonance (NMR) Spectroscopy Kurt Wuthrich Chemistry-2002 Richard Ernst Chemistry...

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