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 (12C, 16O)

Even atomic mass & odd number: I = whole integer (14N, 2H, 10B)

Odd atomic mass: I = half integer (1H, 13C, 15N, 31P)





The spinning nuclei possess angular momentum, P, and charge, and so an associated magnetic moment, .

= x P

Where  is the gyromagnetic ratio

 NMR Spectroscopy
Basic Principles

B0 The spin states of the nucleus are quantified:

I, (I - 1), (I - 2), … , -I

I=  (e.g. 1H)

–
Energy

 – E=h=hB0/2


B0=0 B0>0
NMR Spectroscopy
Basic Principles

In the ground state all nuclear spins are disordered, and

there is no energy difference between them. They are
degenerate.

Since they have a magnetic moment, when we apply a

strong external magnetic field (Bo), they orient either
against or with it:
Bo There is always a small excess of nuclei (population
excess) aligned with the field than pointing against it.
 NMR Spectroscopy
Basic Principles



E=h0=hB0/2
Bo>0
0 is the Larmor Frequency
0=B0, angular velocity
Bo=0


B0 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 = eE/kT

E for 1H at 400 MHz (B0 = 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

1022 600
-rays 500 10 aldehydic
1020 Mossbauer 1H
400 8
19F aromatic
X-rays
1018 300 6
olefinic
4
ultraviolet 1016 electronic 200 acetylenic
31P 2
visible 1014 aliphatic
0
infrared vibrational
1012 13C
/ppm
100
microwave
1010 rotational
 /MHz
108 NMR
radiofrequency
106

 /Hz
 NMR Spectroscopy
The Vector Model

M0

y y

x x

y y

x x
 NMR Spectroscopy
NMR excitation

z
Mo B1 = C * cos (
ot)

y
B1
x
Bo i
Transmitter coil (x)

B1 is an oscillating magnetic field

y y

+0
-0

x x
 NMR Spectroscopy
Laboratory vs. Rotating frame

z z’
+0

M0 M0

y y’
B1
+0

x x’
-0
-20

Laboratory frame Rotating frame

 NMR Spectroscopy
Effect on an rf pulse

z z
=

1tp (degrees)
M0

=90°
y y
B1 B1

x x =180°
 NMR Spectroscopy
Magnetization properties

1H=400,000,000 Hz

A=400,000,005 Hz
z

MAy
y y y
B1 MAx

x

=5 Hz
x x

y y y

x x x
 NMR Spectroscopy
Magnetization properties

1H=400,000,000 Hz

A=400,000,005 Hz
z

MAy
y y y
B1 MAx

x

=5 Hz detector
x x

MAy=MA cos
t MAx=MA sin
t

IM IM
MA MA

t
t t
 NMR Spectroscopy
The Fourier Transform

FT
time domain frequency domain

=1/
t
IM

t
FT

t
frequency
time
 NMR Spectroscopy
The Fourier Transform

IM
Signal Induction Decay (FID)

time
NMR Spectroscopy
The Fourier Transform

FT
 NMR Spectroscopy
Continuous wave vs. pulsed NMR

o or Bo

o or Bo

time
 NMR Spectroscopy
Continuous wave vs. pulsed NMR

•
For cos(t)

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

*
=

tp

Since f=1/t, a pulse of 10
s

duration excites a frequency
bandwidth of 105 Hz!

FT

o
 NMR Spectroscopy
Continuous wave vs. pulsed NMR

E t ~ h or 
t ~1
 NMR Spectroscopy
Single-channel signal detection


FT
y

x +

0

+
-

 NMR Spectroscopy
Quadrature detection

My Mx



y

x +

My Mx
 NMR Spectroscopy
Quadrature detection

cos

y
sin
x +

0

+
-
Hz

+
Hz

0

 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 field 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=hB/2

Nuclei, however, in molecules are never isolated from other particles that are charged
and are in motion (electrons!).

Thus, the field actually felt by a nucleus is slightly different from that of the applied
external magnetic field!!
 NMR Spectroscopy
The Chemical Shift

E=h=hBe/2

Beff, is given by B0-B= B0-B0=B0(1-)

B0(1-)
=
2

(-ref)
and  is the chemical shift
= 106
106 (ref-)
ref
 NMR Spectroscopy
The Chemical Shift
methyl
protons

methylene
amide protons
protons protons
aromatic ring

750 MHz 1H 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 field effect

solvent effect
 NMR Spectroscopy
Nuclear Shielding - diamagnetic contribution

The external field B0 causes the electrons to circulate within their orbitals

B0

h
B0 h
B0(1-)

B’

The higher is the electron density close to the nucleus, the larger the protection is!
 NMR Spectroscopy
Nuclear Shielding - diamagnetic contribution

Depends on the electronegativity

CH3X
 NMR Spectroscopy
Nuclear Shielding - paramagnetic contribution

The external field B0 mixes the wavefunction of the ground state with that of the excited state
The induced current generates a magnetic field that enhances the external field and deshields the
nucleus

LUMO
B0

HOMO

1 1
p = 

R3
 NMR Spectroscopy
Chemical shift range

1H; ~10 ppm

13C; ~200 ppm

19F; ~300 ppm

31P; ~500 ppm

Local diamagnetic and paramagnetic currents make only modest contributions to 1H 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 field.

In isotropic cases:
= (
11 +
22 +
33)

In static cases, e.g. solid state

 NMR Spectroscopy
Nuclear Shielding - neighboring group

B0
B
A

A
B

μpar > μper μpar < μper

- +

+ + - -

- +
 NMR Spectroscopy
Nuclear Shielding - neighboring group

+
- C C - μpar > μper
+

-
+ C C + μpar < μper
-

C 2H 4 C 2H 2 C 2H 6

7 6 5 4 3 2 1 0
ppm
 NMR Spectroscopy
Nuclear Shielding - ring-current effect

More pronounced in aromatic rings due to the
electron clouds

Bo 9.28

e-
-2.99
 NMR Spectroscopy
Nuclear Shielding - hydrogen bonding

Hydrogen bonding causes deshielding due to electron density decrease at the proton site

[EtOH] in CCl4
OH CH2 CH3
1M

0.1M

0.01M

0.001M

6 4 2 0 ppm
 NMR Spectroscopy
Spin-spin (scalar) coupling

HF (1H-19F)

H F

JHF JHF
 NMR Spectroscopy
Spin-spin (scalar) coupling

HF (1H-19F)

H F
H
H F
19F 1H
Bo

H F
H
H F
19F 1H

Nuclear moment

Magnetic polarization
of the electron
E=h JAX mA mX

where m is the magnetic quantum number

JAX is the spin-spin coupling constant
 NMR Spectroscopy
Spin-spin (scalar) coupling

AX2
AMX

AX3
 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 finding the electron at the nucleus (R=0)

Magnitude in Hz- independent of the external magnetic field

H3C – CH3 125 Hz

H2C – CH2 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

3J = A + B cos  + C cos2 

 NMR Spectroscopy
Chemical shifts on the rotating frame

500 MHz
z

y 3 2

0
x

t z

=500 Hz
x
 NMR Spectroscopy
Spin couplings on the rotating frame

J
z

y
0

x

t z

=-J/2 Hz
y

=+J/2 Hz
x
 NMR Spectroscopy
The basic spin-echo pulse sequence

180° applied
90° applied along y axis
along x axis
x y


acquisition

delay
 NMR Spectroscopy 90° applied
along x axis
180° applied
along y axis

x y

Effect of spin echo on chemical shift evolution 

acquisition

delay

z
z

90x
y y

x x
z

z
y
z
180y
A
y
A x
y
x

x
 NMR Spectroscopy 90° applied
along x axis
180° applied
along y axis

x y

Effect of spin echo on scalar coupling evolution 

acquisition

delay

z
z

90x 1H-X
y y

x x
z

z -J/2
y
1H)
z (only
180y +J/2
y
+J/2 x
y
x

-J/2
x
 NMR Spectroscopy 90° applied
along x axis
180° applied
along y axis

x y

Effect of spin echo on scalar coupling evolution 

acquisition

delay

z
z

90x 1H-X
y y

x x
z

z -J/2
y
z (both 1H and X)
180y +J/2
y
-J/2 x
y
x

+J/2
x
 NMR Spectroscopy
Water suppression by the Jump and Return method



z
z

90x
y y


x x
z

z
y
z
A
y
90-x x
y
x

x
NMR Spectroscopy
Water suppression
 NMR Spectroscopy
Spin decoupling

1H decouple

13C

H C
H
H C

H C
H
H C
 NMR Spectroscopy
The J-modulated spin echo

1H decouple

x y



13C
NMR Spectroscopy 1H decouple

x y
The J-modulated spin echo 


13C
 NMR Spectroscopy 1H decouple

x y
The J-modulated spin echo 


13C

If  =180J degrees

C: I=1
CH:

I cos

CH2:

I cos2

CH3:

I cos3

NMR Spectroscopy 1H decouple

x y
The J-modulated spin echo 


13C

=1/J

13C (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
S/N

5/2

Noise increases a square-root of observed frequency

 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.




C2 1H-13C spin pair

H2

H1 

C1



 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


C
2+2C
2C

C2

C2 2
2 H2

+C
H2 +C

H1 

H1 

2
2 
C

C (inverted) C1
C1
2+2C
2C

+C
+C

C1 C2
H1 H2
H1 H2

C1 C2

-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 refocused,

INEPT 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

 NMR Spectroscopy
Longitudinal Relaxation: Establishing Equilibrium

z z z

y y y

x x x

z z

x x
 NMR Spectroscopy
Longitudinal Relaxation: Establishing Equilibrium

Recovery of the z-magnetization follows exponential behavior

dMz (M0-Mz)
= Mz=M0 (1-2e-t/T1)
dt T1

where T1 is the longitudinal relaxation time

 NMR Spectroscopy
Longitudinal Relaxation: Measurement
x x


z z z z

180x 
90x
y y y y
x x x x

z z


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

x x


 NMR Spectroscopy
Longitudinal Relaxation: Exponential growth

Mz=M0 (1-2e-t/T1)

By the end of 5T1 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

- - -
y y y y

+ + +

x x x x

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

1
 =

T2*
 NMR Spectroscopy
Transverse Relaxation: Measurement

x y



z z z

90x 
-

y y y
+ z
x x x
180y

+
z y

-
x

y
x
 NMR Spectroscopy
Transverse Relaxation: Measurement
 NMR Spectroscopy
T1 vs T2 Relaxation

T1
T2

For small molecules, T1  T2

For large molecules, T1 >> T2

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 field fluctuating 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

deflection 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 finding 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 fields fluctuating at the Larmor frequency
0

So, the relaxation rate (R1) will be proportional to the J(
0) 1/T1= R1 =
2 <B2> 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 T1 is minimum (R1 maximum)

0
c<<1 0
c>>1
0
c<<1 (small molecules), J(0) ~ 2
c and T1
decreases (R1 increases) with increasing
c
(e.g.by decreasing the temperature)

0
c=1
0
c>>1 (large molecules), J(0) ~ 2/02
c
and T1 increases (R1 decreases) with
increasing
c (e.g. by decreasing the
temperature)
 NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Nuclei with non-zero quantum numbers have magnetic dipoles

They behave like small magnets and induce small magnetic fields that affect neighboring nuclei

Magnetic field, B
, generated by a magnetic dipole

 NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole

Representation of the dipolar magnetic field B
, generated by a magnetic dipole

lines of force density plots

Bμz Bμx

Bμz is zero for
=±54.7˚ (magic angle)

 NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
The z component of their dipole magnetic field will affect the field experienced by the
other nucleus and cause splitting

X ± sign refers to the quantum number of A (±
)

A
Bμ

Thus, the splitting in the spectrum of X is

A

KAX vary with the distance

e.g. KCH 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 fixed distances and angles the dipolar splitting vary with the crystal
orientation with respect to the external magnetic field
 NMR Spectroscopy
Relaxation mechanisms: Dipole-dipole
Molecules in liquids rotate, “tumble” rapidly with typical frequencies between 1012 to
108 Hz for small molecules and proteins, respectively.
Those frequencies are much larger than typical dipolar couplings (105 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 field experienced at one nucleus as a result of its neighbor will fluctuate 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 efficient 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 field.

As the molecule tumbles, it creates a fluctuating magnetic

field
 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

 NMR Spectroscopy
Nuclear Overhauser Effect (NOE)

Origin of the NOE

N-/2

N-


S 

S I I


0 

N
N
N
/2
N+/2









0
I I 
S

S

N+
N+/2

S I S I
 NMR Spectroscopy
Nuclear Overhauser Effect (NOE)

Six possible transitions in a two-spin system



W1S W1I
W2



W0

W1I W1S



Only single transitions can by observed by NMR (W1)

W0 and W2 are cross-relaxation pathways, responsible for the NOE

 NMR Spectroscopy
Nuclear Overhauser Effect (NOE)

N-
N-/2


S 

S I I


0 

N
N
N
/2
N+/2









0
I 
S I 
S

N+
N+/2

S I S I

S I S I
0
>
0
<

W0
W2
>
0
<
0

I S I S

negative NOE
positive NOE
S I S I
 NMR Spectroscopy
Nuclear Overhauser Effect (NOE)

W1 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)

1H at 400 MHz

W1 at 400 MHz
W2 at 800 MHz (WI+WS)- stimulated by rapidly tumbling molecules
W0 at Hz-kHZ (|WI-WS|)- 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

Bg

Variation of magnetic field strength along the z axis

 NMR Spectroscopy
Field gradient

+!Bg -!Bg

90º Bg, !g -Bg, !g

-!Bg +!B
g

defocused refocused
(dephased) (rephased)
 NMR Spectroscopy
Field gradient

RF

Gz

stronger gradient
 NMR Spectroscopy
Field gradient

RF

Gz

Variation of the second gradient pulse (90 to 110% of the first)

 NMR Spectroscopy
Diffusion-ordered spectroscopy

Bg

!! !!

x x
"! "!
!!
#! #!

G gradient strength

D diffusion coefficient
 NMR Spectroscopy
Diffusion-ordered spectroscopy

mobility
 NMR Spectroscopy
Multi-dimensional NMR

One dimension

Two dimensions
 NMR Spectroscopy
Multi-dimensional NMR

To generate a spectrum with two frequency domains, f1 and f2, it is necessary to sample
data as a function of two separate time variables, t1 and t2.

General scheme for 2D NMR experiment

D
P: Preparation
E: Evolution
M: Mixing P E M
D: Detection
t1 t2
NMR Spectroscopy
Multi-dimensional NMR

hv
A 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 !m
t2
t1
spin-lock

JAB
A B C D E
JBC
A B C D E
!m JCD
A B C D E
JDE
A B C D 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

Relative sensitivity
P E M D 1H-31P 1H-13C 1H-15N

a) H
1 1 1
t1
t2
X

b) H 2.5 4 10
t1

t2 (traditional)
X

c) H
4 8 30
t1
t2
X

d) H
10 32 300
t1 t2
X (inverse)
modern
 NMR Spectroscopy
Heteronuclear Single Quantum Coherence (HSQC)

13C

1H
NMR Spectroscopy
Protein NMR
2D NOESY
NMR Spectroscopy
Protein NMR
2D NOESY
NMR Spectroscopy
Protein NMR
Isotopically labeled proteins
 NMR Spectroscopy
Protein NMR
1H-15N HSQC (protein’s fingerprint)

15N

1H
 NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR

110 ppm

120 ppm

130 ppm

120 ppm 130 ppm 110 ppm

15N 15N 15N

1H 1H 1H
6.5 ppm 6.5 ppm 6.5 ppm
 NMR Spectroscopy
Protein NMR
Signal overlap problem alleviated by 3D & 4D NMR

2D

110 ppm

3D
120 ppm

130 ppm

120 ppm 130 ppm 110 ppm

15N 15N 15N

1H 1H 1H
6.5 ppm 6.5 ppm 6.5 ppm
F2 (15N)

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 C H

N C C

H H O

3D HNCA
 NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
 NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments

15N ppm 115 118 122 125 116 130 128

40

45

13C ppm

50

55

6.5 6.2 7.0 7.2 7.8 8.5 8.0

1H ppm
 NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments
 NMR Spectroscopy
Protein NMR
Assignment - Triple Resonance Experiments

HNCA HN(CO)CA HNCA HN(CO)CA HNCA HN(CO)CA

130 ppm 130 ppm 40 125 ppm 125 ppm 40 125 ppm 125 ppm 40

45 45 45

50 50 50

55 55 55

8.5 8.5 7.5 7.5 7.5 7.5

i-1 i i+1