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BSB 512
Nuclear Magnetic Resonance Spectroscopy
TTh 2:20 - 3:25 pm
Lecture notes available at http://sos.bio.sunysb.edu/bsb512
Lecture
Lecture
Lecture
Lecture
1:
2-4:
5:
6:
Basics
Protein structure determination
Relaxation and dynamics
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.
Levitt, MH
Cavanagh J. et al.
Ernst, R. et al.
Bax, A
NMR of Proteins and Nucleic Acids
Spin Dynamics
Protein NMR Spectroscopy
Principles of NMR in One and Two Dimensions
Two dimensional NMR in Liquids
NMR Spectroscopy: Some history
1915
1922
1946
1967
1971
1976
1986
Einstein and de Hass - Correlation between magnetic moment and
spin angular momentum
Stern and Gerlach - Spins are quantized
Bloch and Purcell - First NMR experiment
Richard Ernst
- Fourier transformations
Jean Jeener
- Two dimensional NMR - COSY
Richard Ernst
- First two dimensional NMR experiment
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
N
N
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
Bo
H
H C H
N
H
C C
H
Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space)
H
H C H
N C C
H H
H
H C H
N C C
H H
H
H C H
N C C
H H
H
H C H
N C C
H H
Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space)
3D HSQC - NOESY for
Inter-residue contacts
Magnetization can be
transferred between 1H, 13C and
15N to establish connectivities
HNCA
HNCOCA
HNCOCACB etc
HSQC-TOCSY
They all use INEPT tranfers
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
ee-
Concept 5: When the B1 field is turned on, the net magnetization
rotates down into the XY plane
z
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
y=e
-x/t
x
y
y = 1- e -x/t
x
Mz
Mz = Mo (1- e
-t/T1
)
t
Concept 7: Individual spins precess about the magnetic field axis.
z
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
y
x
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
y
x
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).
has a large chemical shift range (300 ppm).
113Cd
What is a ppm? Ppm = part per million
400 MHz
100 MHz
1H
13C
30 MHz
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.
b
b
CH3
a
a
C-OH
C-OH
CH3
frequency
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.)
y
x
e-
e-
b
100,010,000 Hz
b
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.
y
reference frequency
-5,000 Hz below
reference
CH3
C-OH
CH3
x
C-OH
+5,000 Hz above
reference
frequency
Chemical Shifts
J-couplings (through bond)
Dipolar couplings (through space)
Structure
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
that overlap in the time domain
Fourier Transform
t1 -> f1
f1
General Two Dimensional Experiment
p/2
p/2
t
Fourier Transform
t1 -> f1 and t2 -> f2
Acquire
t2
1
f1
f2
General Two Dimensional Experiment
p/2 p/2
t
p/2
Vary t1
Collect a series of 1D spectra
1p/2
t
1
p/2
t
1
Acquire
t2
Acquire
t2
p/2
Acquire
t2
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
1H
chemical shift
Dipolar
coupling
1H
chemical shift
13C
chemical
shift
1H
chemical shift
1H
chemical
shift
1H
chemical shift