NMR Spectroscopy
Applications
Drug design
MRI
Food quality
Metabonomics Structural biology

NMR Spectroscopy
Basic Principles
N.M.R. = Nuclear Magnetic Resonance
Spectroscopic technique, thus relies on the interaction between material and electromagnetic radiation
The nuclei of all atoms possess a nuclear quantum number, I . (I 0, always multiples of .)
Only nuclei with spin number (I) >0 can absorb/emit electromagnetic radiation.
Even atomic mass & number: I = 0 ( 12 C, 16 O)
Even atomic mass & odd number: I = whole integer ( 14 N, 2 H, 10 B)
Odd atomic mass: I = half integer ( 1 H, 13 C, 15 N, 31 P)
The spinning nuclei possess angular momentum, P, and charge, and so an associated magnetic moment, .
= x P
Where is the gyromagnetic ratio

B
0
NMR Spectroscopy
Basic Principles
The spin states of the nucleus are quanti fi ed:
I, (I - 1), (I - 2), … , -I
I= (e.g. 1 H)
B
0
=0
–
B
0
>0
–
E=h =h B
0
/2

NMR Spectroscopy
Basic Principles
In the ground state all nuclear spins are disordered, and there is no energy difference between them. They are degenerate .
B o
Since they have a magnetic moment, when we apply a strong external magnetic fi eld (Bo), they orient either against or with it:
There is always a small excess of nuclei ( population excess) aligned with the fi eld than pointing against it.

B o
=0
B
0
NMR Spectroscopy
Basic Principles
B o
>0
E=h
0
=h B
0
/2
0 is the Larmor Frequency
0
= B
0
, angular velocity z y x

NMR Spectroscopy
Basic Principles
Each level has a different population (N) , and the difference between the two is related to the energy difference by the Boltzmman distribution:
N /N = e E/kT
E for 1 H at 400 MHz (B
0
= 9.5 T) is 3.8 x 10 -5 Kcal/mol
N /N =
1.000064
The surplus population is small (especially when compared to UV or IR).
That renders NMR a rather insensitive technique!

NMR Spectroscopy
The electromagnetic spectrum
10 22
-rays
10 20
X-rays
10 18 ultraviolet 10 16 visible
10 14 infrared
10 12 microwave
10 10 radiofrequency
10 8
10 6
Mossbauer electronic vibrational rotational
NMR
/Hz
600
500
400
300
1 H
19 F
200
31 P
100
13 C
/MHz
10
8
6
4
2
0 aldehydic aromatic olefinic acetylenic aliphatic
/ppm

x x
NMR Spectroscopy
The Vector Model y x
M
0 y y y x

NMR Spectroscopy
NMR excitation
B o x
M o z
B
1 x i
Transmitter coil (x) y
B
1
= C * cos ( o t) y
+
0 x y
-
0
B
1
is an oscillating magnetic field

z
M
0
+
0 x
-
0
Laboratory frame y
NMR Spectroscopy
Laboratory vs . Rotating frame z’
M
0 x’
B
1
-2
0
Rotating frame y’
+
0

x
B
1 z
M
0
NMR Spectroscopy
Effect on an rf pulse y x
B
1 z
=
1 t p
(degrees) y
=90
°
=180
°

x
B
1 z
A y
NMR Spectroscopy
Magnetization properties
1
H
A
=400,000,000 Hz
=400,000,005 Hz x y
=5
Hz
M
Ax
M
Ay y x x y x y x y

z
A y x
B
1
I
M
M
A
M
Ay
=M
A
cos t
NMR Spectroscopy
Magnetization properties
1
H
A
=400,000,000 Hz
=400,000,005 Hz x
I
M
M
A t t y
=5
Hz
M
Ax
M
Ay y detector x
M
Ax
=M
A
sin t t

I
M
NMR Spectroscopy
The Fourier Transform time domain
FT frequency domain
=1/ t t
FT t time frequency

I
M
NMR Spectroscopy
The Fourier Transform
Signal Induction Decay (FID) time

NMR Spectroscopy
The Fourier Transform
FT

o
or B o
NMR Spectroscopy
Continuous wave vs . pulsed NMR time o
or B o

• For cos( t)
NMR Spectroscopy
Continuous wave vs . pulsed NMR
FT absorptive lines
• For sin( t)
FT despersive lines

NMR Spectroscopy
Continuous wave vs . pulsed NMR
A monochromatic radiofrequency pulse is a combination of a wave
(cosine) of frequency
0
and a step function
* t p
=
Since f=1/t, a pulse of 10 s duration excites a frequency bandwidth of 10 5 Hz !
FT o

NMR Spectroscopy
Continuous wave vs . pulsed NMR
E t ~ h or t ~1

NMR Spectroscopy
Single-channel signal detection x z
+ y
FT
+
0
-

x
NMR Spectroscopy
Quadrature detection z
+ y
My
My
Mx
Mx

x
+ y
NMR Spectroscopy
Quadrature detection cos sin
+
0
Hz
+
0
Hz

NMR Spectroscopy
The Chemical Shift
The NMR frequency of a nucleus in a molecule is mainly determined by its gyromagnetic ratio and the strength of the magnetic fi eld B
The exact value of depends, however, on the position of the nucleus in the molecule or more precisely on the local electron distribution
this effect is called the chemical shift

NMR Spectroscopy
The Chemical Shift
E=h =h B/2
Nuclei, however, in molecules are never isolated from other particles that are charged and are in motion (electrons!).
Thus, the fi eld actually felt by a nucleus is slightly different from that of the applied external magnetic fi eld!!

and is the chemical shift
NMR Spectroscopy
The Chemical Shift
E=h =h B e
/2
B eff
, is given by B
0
-B = B
0
-B
0
= B
0
(1)
=
B
0
(1)
2
=
(
ref ref
)
10 6 10 6 ( ref
)

NMR Spectroscopy
The Chemical Shift amide protons aromatic ring
protons methylene protons methyl protons
750 MHz 1 H spectrum of a small protein shielding frequency magnetic field

NMR Spectroscopy
The Chemical Shift

NMR Spectroscopy
The Chemical Shift

NMR Spectroscopy
Nuclear Shielding
= dia
+ para
+ nb
+ rc
+ ef
+ solv diamagnetic contribution paramagnetic contribution neighbor anisotropy effect ring-current effect electric fi eld effect solvent effect

NMR Spectroscopy
Nuclear Shielding - diamagnetic contribution
The external fi eld B
0
causes the electrons to circulate within their orbitals
B
0 h B
0 h B
0
(1)
B’
The higher is the electron density close to the nucleus, the larger the protection is!

CH
3
X
NMR Spectroscopy
Nuclear Shielding - diamagnetic contribution
Depends on the electronegativity

NMR Spectroscopy
Nuclear Shielding - paramagnetic contribution
The external fi eld B
0
mixes the wavefunction of the ground state with that of the excited state
The induced current generates a magnetic fi eld that enhances the external fi eld and deshields the nucleus
LUMO
HOMO
B
0 p
=
1 1
R 3

NMR Spectroscopy
Chemical shift range
1 H; ~10 ppm
13 C; ~200 ppm
19 F; ~300 ppm
31 P; ~500 ppm
Local diamagnetic and paramagnetic currents make only modest contributions to 1 H shielding!

NMR Spectroscopy
Chemical Shift Anisotropy
Nuclear shielding, , is a tensor .
The distribution of the electrons about the nucleus is non-sperical- thus, the magnitude of the shielding depends on the relative orientation of the nucleus with respect to the static fi eld.
In isotropic cases: = (
11
+
22
+
33
)
In static cases, e.g. solid state

B
0
NMR Spectroscopy
Nuclear Shielding - neighboring group
A
B
A
B
μ par
>
μ per
+
-
-
+
μ par
<
μ per
-
+
-
+

NMR Spectroscopy
Nuclear Shielding - neighboring group
- C
+
C -
+
+
-
C
-
C
+
μ par
>
μ per
μ par
<
μ per
C
2
H
4
C
2
H
2
C
2
H
6
7 6 5 4 3 2 1 0 ppm

B o e -
NMR Spectroscopy
Nuclear Shielding - ring-current effect
More pronounced in aromatic rings due to the electron clouds
-2.99
9.28

NMR Spectroscopy
Nuclear Shielding - hydrogen bonding
Hydrogen bonding causes deshielding due to electron density decrease at the proton site
OH CH
2
CH
3
[EtOH] in CCl
4
1M
0.1M
0.01M
0.001M
6 4 2 0 ppm

H
J
HF
NMR Spectroscopy
Spin-spin (scalar) coupling
HF ( 1 H 19 F )
F
J
HF

B o
19 F
19 F
Nuclear moment
Magnetic polarization of the electron
NMR Spectroscopy
Spin-spin (scalar) coupling
HF ( 1 H 19 F )
H F
1 H
H
H F
H F
H F
H
1 H
E=h J
AX
m
A
m
X where m is the magnetic quantum number
J
AX
is the spin-spin coupling constant

AMX
NMR Spectroscopy
Spin-spin (scalar) coupling
AX
2
AX
3

NMR Spectroscopy
Spin-spin (scalar) coupling
Strong coupling – <10|J|

NMR Spectroscopy
Spin-spin (scalar) coupling
The principal source of scalar coupling is an indirect interaction mediated by electrons involved in chemical bonding
The magnitude of interaction is proportional to the probability of fi nding the electron at the nucleus (R=0)
Magnitude in Hz - independent of the external magnetic fi eld
H
3
C – CH
3
125 Hz
H
2
C – CH
2
160 Hz
HC CH 250 Hz

NMR Spectroscopy
Spin-spin (scalar) coupling
Three-bond coupling most useful since it carries information on dihedral angles
Empirical relationship: the Karplus relation
3 J = A + B cos + C cos 2

x z
NMR Spectroscopy
Chemical shifts on the rotating frame y t z x
=500 Hz y
3 2
0
500 MHz

x z
NMR Spectroscopy
Spin couplings on the rotating frame
J y t z x
=-J/2 Hz y
=+J/2 Hz
0

NMR Spectroscopy
The basic spin-echo pulse sequence
90° applied along x axis x delay
180° applied along y axis y acquisition

x x z z
NMR Spectroscopy
90° applied
Effect of spin echo on chemical shift evolution along x axis x delay
180° applied along y axis y z acquisition y
90 x y x z y y z
180 y
A y x
A x

x x z z
NMR Spectroscopy
90° applied
Effect of spin echo on scalar coupling evolution along x axis x delay
180° applied along y axis y z acquisition y
90 x
1 H-X y x z
-J/2 y
+J/2 y z
-J/2
+J/2
(only 1 H)
180 y y x x

x x z z
NMR Spectroscopy
90° applied
Effect of spin echo on scalar coupling evolution along x axis x delay
180° applied along y axis y z acquisition y
90 x
1 H-X y x z
-J/2 y
+J/2 y z
+J/2
(both 1 H and X)
180 y
-J/2 y x x

x x z z
NMR Spectroscopy
Water suppression by the Jump and Return method z y
90 x x y z z y
90
-x y x x
A y

NMR Spectroscopy
Water suppression

1 H
13 C
NMR Spectroscopy
Spin decoupling decouple
H C
H
H C
H C
H C
H

NMR Spectroscopy
The J-modulated spin echo
1 H
13 C x y decouple

NMR Spectroscopy
The J-modulated spin echo
1 H
13 C x y decouple

If =180J degrees
NMR Spectroscopy
The J-modulated spin echo
1 H
13 C x y decouple
C: I=1
CH: I cos
CH
2
: I cos 2
CH
3
: I cos 3

NMR Spectroscopy
The J-modulated spin echo
1 H
13 C x y decouple
=1/J
13 C (ppm)

NMR Spectroscopy
Sensitivity enhancement
NMR has poor sensitivity compared to other analytical techniques
The intrinsic sensitivity depends upon the gyromagnetic ratio,
A greater contributes to: a high resonant frequency- large transition energy difference- greater Boltzmann population difference high magnetic moment and hence a stronger signal high rate of precession which induces a greater signal in the detection coil
So, the strength of NMR signal is proportional to 3
Noise increases a square-root of observed frequency
}
S/N 5/2

NMR Spectroscopy
Sensitivity enhancement by polarization transfer
Signal sensitivity enhancement by transferring the greater population differences of high-
spins onto their spin-coupled low partners.
C 2
H 2
H 1
C 1
1 H13 C spin pair

NMR Spectroscopy
Sensitivity enhancement by polarization transfer
Signal sensitivity enhancement by transferring the greater population differences of high-
spins onto their spin-coupled low partners.
2 C
C 2
H 2
2
+ C
2
H 1
+ C
C 1
2 C
C
H 1 H 2
C 1 C 2
C
C
2 +2 C
C 2
+ C
2
(inverted)
H 1
H 2
2
C 1
2 +2 C
+ C
C 1 C 2
H 1 H 2
C
-3:5

NMR Spectroscopy
Sensitivity enhancement by polarization transfer
Signal sensitivity enhancement by transferring the greater population differences of high-
spins onto their spin-coupled low partners.
coupled decoupled INEPT refocused
INEPT refocused, decoupled
INEPT

NMR Spectroscopy
Relaxation
When perturbed, the nuclear spins need to relax to return to their equilibrium distribution
E.g. when the sample is put into a magnet, the Boltzmann distribution of spins among the energy levels changes due to a change in the energy of the various levels
E.g. after applying electromagnetic radiation, which induces transitions between energy levels, the system returns to its equilibrium
This process is called relaxation

x z
NMR Spectroscopy
Longitudinal Relaxation: Establishing Equilibrium z z y y x x z z y x x

NMR Spectroscopy
Longitudinal Relaxation: Establishing Equilibrium
Recovery of the z-magnetization follows exponential behavior dM z
(M
0
=
dt T
1
-M z
)
M z
=M
0
(1-2e -t/T1 where T
1
is the longitudinal relaxation time
)

NMR Spectroscopy
Longitudinal Relaxation: Measurement x x x z y
180 x x z y x z y
90 x x z y z z x y
90 x x y

NMR Spectroscopy
Longitudinal Relaxation: Measurement x x

NMR Spectroscopy
Longitudinal Relaxation: Exponential growth
M z
=M
0
(1-2e -t/T1 )
By the end of 5T
1
sec, the magnetization has recovered by 99.33%

NMR Spectroscopy
Longitudinal Relaxation: optimizing sensitivity

NMR Spectroscopy
Longitudinal Relaxation: optimizing sensitivity

NMR Spectroscopy
Longitudinal Relaxation: optimizing sensitivity optimum pulse repetition time when using 90º
Quantitative measurements and integration

NMR Spectroscopy
Transverse Relaxation: magnetization loss in the x-y plane x y x
- y
+ time x
- y
+ x
- y
+

NMR Spectroscopy
Transverse Relaxation: magnetization loss in the x-y plane
=
1
T
2
*

NMR Spectroscopy
Transverse Relaxation: Measurement x y x z y
90 x x z y z x z
-
+ y
180 y z x y x
- y

NMR Spectroscopy
Transverse Relaxation: Measurement

NMR Spectroscopy
T1 vs T2 Relaxation
T
1
T
2
For small molecules, T
1
T
2
For large molecules, T
1
>> T
2
Longitudinal relaxation causes loss of energy from the spins (enthalpic)
Transverse relaxation occurs by mutual swapping of energy between spins (entropic)

NMR Spectroscopy
Relaxation mechanisms
Nuclear spin relaxation is not a spontaneous process; it requires stimulation by suitable fluctuating fields to induce the necessary spin transitions
Two main mechanisms
Dipole-dipole
Chemical shift anisotropy

NMR Spectroscopy
Relaxation mechanisms
Longitudinal relaxation requires a time-dependent magnetic fi eld fl uctuating at the Larmor frequency
The time-dependence originates in the motions of the molecule (vibration, rotation, diffusion etc)
Molecules in solution “ tumble ”. This “tumbling” can be characterized by a rotational correlation time c c
is the time needed for the rms de fl ection of the molecules to be ~ 1 radian (60°)

NMR Spectroscopy
Spectral density function
Rotational diffusion in solution occurs at a range of frequencies
1/ c
~ rms rotational frequency (radians s -1 )
The probability function of fi nding motions at a given angular frequency can be described by the spectral density function J( )

NMR Spectroscopy
Spectral density function
Frequency distribution of the fluctuating magnetic fields

NMR Spectroscopy
Spectral density function: Longitudinal relaxation
Spins are relaxed by local fi elds fl uctuating at the Larmor frequency
0
So, the relaxation rate (R1) will be proportional to the J(
0
) 1/T
1
= R
1
= 2 <B 2 > J(
0
)
Knowing the form of J( ) we can predict the dependence of the spin-lattice relaxation time (T1=1/
R1) on the correlation time c
for a given NMR frequency
0
0 c
=1 ,J(
0
) = c
= 1/
0 and T
1
is minimum (R
1
maximum)
0 c
<<1 (small molecules), J( decreases (R
1
0
) ~ 2
increases) with increasing
(e.g.by decreasing the temperature) c
and T
1 c
0 c
>>1 (large molecules), J(
0
) ~ 2/ and T
1
increases (R
1
decreases) with
0
2 increasing c
(e.g. by decreasing the temperature) c
0 c
<<1
0 c
=1
0 c
>>1

NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Nuclei with non-zero quantum numbers have magnetic dipoles
They behave like small magnets and induce small magnetic fi elds that affect neighboring nuclei
Magnetic fi eld, B , generated by a magnetic dipole

NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Representation of the dipolar magnetic fi eld B , generated by a magnetic dipole lines of force density plots
B
μ z
B
μ z
is zero for =±54.7˚ (magic angle)
B
μ x

NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
The z component of their dipole magnetic fi eld will affect the fi eld experienced by the other nucleus and cause splitting
± sign refers to the quantum number of A (± ) X
B
A
μ
Thus, the splitting in the spectrum of X is
A
K
AX
vary with the distance e.g. K
CH
is 9000 Hz at 1.5 Å and 30 Hz at 10 Å

NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Splitting of the AX spectrum depends on
In a crystal with fi xed distances and angles the dipolar splitting vary with the crystal orientation with respect to the external magnetic fi eld

NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Molecules in liquids rotate, “tumble” rapidly with typical frequencies between 10 12 to
10 8 Hz for small molecules and proteins, respectively.
Those frequencies are much larger than typical dipolar couplings (10 5 Hz)
The angular part of the dipolar splitting is averaged over all possible orientation to 0
Although they are not directly observed in solution, dipolar couplings play an important role in spin relaxation
The local fi eld experienced at one nucleus as a result of its neighbor will fl uctuate as the molecule tumbles

NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
R1 depend of the gyromagnetic ratio of the nuclei (e.g. H-H relaxation more ef fi cient than C-H)

NMR Spectroscopy
Relaxation mechanisms: Chemical shift anisotropy
The distribution of the electrons about the nucleus is non-sperical- thus, the magnitude of the shielding depends on the relative orientation of the nucleus with respect to the static fi eld.
As the molecule tumbles, it creates a fl uctuating magnetic fi eld

NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
NOE : change in intensity of one resonance when the spin transitions of another are perturbed from their equilibrium populations perturbation : saturation or inversion
The two spins should “ communicate ” through dipole-dipole interaction
NOE is observed for spin I when spin S is perturbed

N
S
N-
I
N
I S
N+
S I
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
Origin of the NOE
N /2
S
0
N/2
I
N+ /2
I
0
S
N+ /2
S
I

NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
Six possible transitions in a two-spin system
W
1
S
W
0
W
2
W
1
I
W
1
I
W
1
S
Only single transitions can by observed by NMR (W1)
W
0
and W
2
are cross-relaxation pathways, responsible for the NOE

N
S
I
N-
N+
NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
I
N
S
N /2
S
0
N/2
I
N+ /2
I
0
S
N+ /2
S I
S
I
S
0
I
>
I
>
W
2
0
S
S
0
W
0
<
I
<
I
0
S
S I positive NOE
S I negative NOE

NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
W
1 tends to reduce the magnitude of the NOE
Saturating for a period of time that is long relative to the relaxation times allows a new steady-state of populations to arise
IS
, cross-relaxation rate: dictates the sign of the NOE
IS
, dipolar longitudinal relaxation rate of spin I: it serves to reduce the magnitude
Thus, NOE is related to molecular motion!

NMR Spectroscopy
Nuclear Overhauser Effect (NOE)
1 H at 400 MHz
W
1
at 400 MHz
W
2
at 800 MHz ( W
I
+W
S
)- stimulated by rapidly tumbling molecules
W
0
at Hz-kHZ (| W
I
-W
S
| )- stimulated by slowly tumbling molecules
Small molecules exhibit positive NOEs
Large molecules exhibit negative NOEs

NMR Spectroscopy
Nuclear Overhauser Effect (NOE) c
=1.12
Variation in NOE as a function of molecular tumbling rates

NMR Spectroscopy
Field gradient
B g
Variation of magnetic field strength along the z axis

90º
NMR Spectroscopy
Field gradient
+ !
B g
!
B g
B g
, !
g
-B g
, !
g
!
B g defocused
(dephased)
+ !
B g refocused
(rephased)

RF
NMR Spectroscopy
Field gradient x
G z stronger gradient

RF
G z
NMR Spectroscopy
Field gradient x
Variation of the second gradient pulse (90 to 110% of the first)

NMR Spectroscopy
Diffusion-ordered spectroscopy
B g
!
!
!
!
x
# !
" !
!
!
x
# !
" !
G gradient strength
D diffusion coefficient

NMR Spectroscopy
Diffusion-ordered spectroscopy mobility

One dimension
Two dimensions
NMR Spectroscopy
Multi-dimensional NMR

NMR Spectroscopy
Multi-dimensional NMR
To generate a spectrum with two frequency domains , f
1
and f data as a function of two separate time variables , t
1
and t
2
.
2
, it is necessary to sample
P: Preparation
E: Evolution
M: Mixing
D: Detection
General scheme for 2D NMR experiment
P E t
1
M
D t
2

NMR Spectroscopy
Multi-dimensional NMR
A hv
B

NMR Spectroscopy
Multi-dimensional NMR

NMR Spectroscopy
COSY (COrrelated SpectroscopY)
Correlation through bonds (J-coupling)

NMR Spectroscopy
TOCSY (Total COrrelated SpectroscopY)
Correlation through bonds (J-coupling) x t
1
!
m spin-lock t
2
!
m
A
J
AB
A
B
B
J
BC
C
A
A
B
B
D E
C
C
J
CD
D
C
E
D
D
J
DE
E
E

NMR Spectroscopy
COSY vs. TOCSY
Correlation through bonds (J-coupling)
COSY
TOCSY

NMR Spectroscopy
COSY vs. TOCSY
Correlation through bonds (J-coupling)

NMR Spectroscopy
General schemes for 2D NMR a) H b)
X
H c)
X
H
X d) H
X
P E t
1
M t t t
1
1
1 t
2 t t t
D
2
2
2
Relative sensitivity
1 H31 P 1 H13 C 1 H15 N
1 1 1
2.5 4 10
(traditional)
4 8 30
10 32 300
(inverse)
modern

NMR Spectroscopy
Heteronuclear Single Quantum Coherence (HSQC)
13 C
1 H

NMR Spectroscopy
Protein NMR
2D NOESY

NMR Spectroscopy
Protein NMR
2D NOESY

NMR Spectroscopy
Protein NMR
Isotopically labeled proteins

NMR Spectroscopy
Protein NMR
1 H15 N HSQC (protein’s fingerprint)
15 N
1 H

NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR
120 ppm
15 N
1 H
6.5 ppm
130 ppm
15 N
1 H
6.5 ppm
110 ppm
120 ppm
130 ppm
110 ppm
15 N
1 H
6.5 ppm

NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR
2D
120 ppm
15 N
1 H
6.5 ppm
130 ppm
15 N
1 H
6.5 ppm
110 ppm
120 ppm
130 ppm
110 ppm
15 N
1 H
6.5 ppm
3D
F2 ( 15 N)
F3 (NH)

NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR

NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR

NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
H
N
C H
C C
H H O
3D HNCA

NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments

NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
15 N ppm
115 118 122 125 116 130 128
40
45
13 C ppm
50
55
6.5 6.2 7.0 7.2
1 H ppm
7.8 8.5 8.0

NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments

NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
HNCA HN(CO)CA
130 ppm 130 ppm
40
HNCA HN(CO)CA
125 ppm 125 ppm
40
HNCA HN(CO)CA
125 ppm 125 ppm
40
45 45 45
50
55
50
55
50
55
8.5 i-1
8.5 7.5 7.5 i
7.5 i+1
7.5