BSB 512

Nuclear Magnetic Resonance Spectroscopy

TTh 2:20 - 3:25 pm

Lecture notes available at http://sos.bio.sunysb.edu/bsb512

Lecture 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.
References

http://www.cis.rit.edu/htbooks/nmr

Books

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
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
1967 Richard Ernst - Fourier transformations
1971 Jean Jeener - Two dimensional NMR - COSY
1976 Richard Ernst - First two dimensional NMR experiment
1986 Kurt Wuthrich - First independent NMR - X-ray comparison
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.

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.
 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.

Bo • These small magnets interact with

each other.

S
13C and 15N also have spin angular momentum and “interact” with 1H
 Magnetization can be
transferred between 1H, 13C and
15N to establish connectivities

H
Bo H C H
C C
N
H
H

Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space)
 H
H C H H
H C H
N C C
3D HSQC - NOESY for

N C C
Inter-residue contacts

H H
H H

H
H C H H Magnetization can be
H C H transferred between 1H, 13C and

N C C
15N to establish connectivities

N C C
H H
HNCA

H H
HNCOCA
HNCOCACB etc
HSQC-TOCSY
Chemical Shifts
J-couplings (through bond) They all use INEPT tranfers
Dipolar couplings (through space)
 Concept 1: Some nuclei have non-zero spin quantum numbers.

e-

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

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-
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

QM picture Classical picture

 Large external magnet

Net magnetization
aligned along Z-axis
of the magnetic field

Bo
The B1 field is produced by a
small coil in the NMR
probe which is placed
in the bore of the large
external magnet.

B1

Bo
Net magnetization
aligned along x-axis
of the magnetic field
after application of B1
field.
NMR magnet.

B1

Bo

NMR probe
e-

e-
Concept 5: When the B1 field is turned on, the net magnetization
rotates down into the XY plane

Bo

x
y
Concept 6: When the B1 field is turned off, the net magnetization
relaxes back to the Z axis with the time constant T1

z
T1
Bo

x
y

T1 is the “longitudinal” relaxation time constant

which results from “spin-lattice” relaxation
Exponential Functions

y=e -x/t

y = 1- e -x/t

Mz

Mz = Mo (1- e -t/T1 )

t
Concept 7: Individual spins precess about the magnetic field axis.

Bo

x
y

Precession frequency = Larmor frequency

wo = -g Bo (MHz)
 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
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
Concept 10: NMR signal is a Fourier transform of the oscillating
current induced in the sample coil

x
y -y y
-x

time

frequency
Chemical Shifts
Concept 12: Nuclear spins produce small magnetic fields
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).
 What is a ppm? Ppm = part per million

400 MHz 100 MHz 30 MHz

1H 13C 15N

1 ppm = 400 Hz
15 ppm = 6000 Hz
15 ppm

1H has a small chemical shift range (15 ppm).

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.

C-OH CH3
b
b

a a
frequency
C-OH CH3
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.)

e- e-
 b
b
100,010,000 Hz 100,000,000 Hz

a a

C-OH CH3

Reference or carrier = 100,005,000 Hz

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
y -5,000 Hz below
reference
C-OH CH3
CH3

C-OH frequency

+5,000 Hz above
reference
Chemical Shifts
J-couplings (through bond) Structure
Dipolar couplings (through space)

H
H C H
C
N C
H H

T1 relaxation
T2 relaxation Dynamics
General One Dimensional Experiment

p/2 Acquire

t
1

Fourier Transform
t1 -> f1

f1
General One Dimensional Experiment

p/2 Acquire

t
1

Fourier Transform
t1 -> f1

f1
 General One Dimensional Experiment

p/2 Acquire

t
1

Fourier Transformation
resolves multiple frequencies Fourier Transform
that overlap in the time domain t1 -> f1

f1
 General Two Dimensional Experiment

p/2 p/2
Acquire

t t2
1
Fourier Transform
t1 -> f1 and t2 -> f2

f1

f2
 General Two Dimensional Experiment

p/2 p/2
Acquire

t t2
p/2 1p/2
Vary t1 Acquire
Collect a series of 1D spectra

t t2
1
p/2 p/2
Acquire

t t2
1
 General Two Dimensional Experiment

t
1

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
 General Two Dimensional Experiment

t
1

Vary t1
Collect a series of 1D spectra

f2

Whereas here, the intensity of is modulated as

a function of the t1 evolution time
 General Two Dimensional Experiment

t
1

Transpose and then

Fourier transform in t1 dimension

f2
 General Two Dimensional Experiment

t
1

Transpose and then

Fourier transform in t1 dimension

f2
 General Two Dimensional Experiment

Projection on f2 gives
original chemical shifts

f1

f2
General Two Dimensional Experiment

Projection on f1 yields
new information

f1

f2
 General Two Dimensional Experiment

J coupling
Dipolar
coupling

1H chemical shift 1H chemical shift

13Cchemical 1H chemical
shift shift

1H chemical shift 1H chemical shift