2D NMR:
Applications
“IUPAB sponsored Workshop on NMR & its
Applications in Biological Systems”
November 23-30, 2009
Mamata Joshi
TIFR, Mumbai
What is 2D NMR?
When and Why?
How to apply?
WHAT?
• Stack of several 1D
spectra
• Each 1D is different from
the next by a small
change in the evolution
time t1
• Parameters for each
successive experiment
in the series are
constant except the
phase of the pulses
• FT of the two time
domains provides a map
of spin-spin correlations
2D spectrum – actual view
Basic features of 2D spectra
HA HB
HA HB
diagonal peak (F1=F2)
H chemical shift (ppm)
crosspeak:
correlation of two
different resonances
by short interatomic
distance or through-bond
1
connection
H chemical shift (ppm)
1
WHEN?
Overlap of signals in the 1D spectrum
No overlap, easy to assign
WHY?
What information are you seeking? Corelation?
• Characterisation/assignments of resonances in the molecule?
• Orientation of different groups/segments of the molecule in
space/ w.r.t. one another (stereochemistry)?
Two main types of homonuclear 2D techniques
Scalar coupling (J) based NOE based
• NOESY- Nuclear Overhauser
• COSY -COrelation SpectroscopY
Effect SpectroscopY
• TOCSY- TOtal Corelation • ROESY- ROtating framE
SpectroscopY SpectroscopY
• MQF- Multiple Quantum Filter
spectroscopy
2D-NMR sequences contain two types
of time periods:
evolution time and mixing time
evolution time: mixing time:
a second frequency magnetisation is
dimension is created by transferred from spin to
indirect detection spin via interactions
between spins
J-correlation based 2D experiments
• Facilitated by electrons in
bonds separating nuclei
• Interaction results in
splitting of resonance
lines in 1D spectrum or
cross peaks in 2D
• Magnitude of J coupling
is dictated by torsion
angles between two
coupling nuclei given by
the Karplus equation
J = A + BCosθ+ CCos2θ
A, B & C are empirical
constants
COSY
• magnitude mode
• upto three-bond coupling
network seen
• ideal for small molecules with
simple coupling networks
• easy processing, no phase
correction
• adequate if only coupling
network has to be established
C 3H 8O
CH3 – CH2 – CH2 – OH
CH2 OH CH2 CH3
C3H8O: COSY
CH3
CH2 OH
CH3 – CH2 – CH2 – OH CH2
DQF COSY
• Phase-sensitive, pure absorption
lineshapes with +ve & -ve
components
• Higher resolution
• J values can be determined
• Less pronounced diagonal ridge,
so easy to assign cross peaks
close to diagonal
• Elimination of strong (solvent)
signals not involved in
homonuclear J-coupling
COSY Vs DQFCOSY
TOCSY
• Cross peaks generated between all members
of a coupled spin network due to
magnetisation transfer from one spin to
another even without direct coupling
• Pure absorption mode spectra with positive
intensity peaks created
• Ideal for assignment of spins in biomolecules
(polypeptides/nucleotides) since
characteristic peak patterns rendered for
specific spin systems.
Other COSY-type experiments
• COSYLR
Long range coupling
networks probed J-Resolve
• HOMO2DJ-Resolve
renders J directly on the F1-
axis of the 2D plane with δ on
the F2-axis
• ECOSY
simplifies complex splitting
patterns by retaining only
direct couplings & eliminating
signals due to passive
coupling
NOE-based experiments
NOESY
• Useful to identify spins
undergoing cross-relaxation
• Direct dipolar couplings provide
primary means of cross-
relaxation, & so spins undergoing
this are close to one another in
space (<5ºA)
• Intensity of peaks depends on
parameter, τm, the mixing time
• Indicated in the form of cross
peaks in the NOESY spectrum
Mixing time τm NOESY
• For small molecules τm ~ T1
• For large molecules low τm is
recommended to avoid spin-
diffusion effects
• For medium sized molecules
(MW about 500-1000), NOE =
0 in many cases. ROESY is the
solution!
• NOE α 1/r6, cross-peak
intensity can be used as a
measure of distance between
the two protons
NOESY applications
Which isomer is responsible for this spectrum?
NOESY
H4
H3
OMe
H5
NMR of Biomolecules
• NMR is the only method for
residue level structure
determination of
biomacromolecules in
aqueous solutions at near
physiological conditions
• Depending on the size of the
molecule, one can go for
homunuclear/heteronuclear
2D corelation or even 3D or
4D!
Dynein light chain protein (DLC8)
89 residues, MW~10.3kD
Structure Determination Strategy for
Polypeptides
• Identify resonances for each amino acid
• Put the assigned resonances in order according to
their amino acid sequence i.e. sequential assignment
• (R-G-S, T-L-G-S)
• Now do the sequence-specific assignment
• Secondary structure determination using NOESY,
(short distances), temperature coefficients
delineation of H-bonded amide protons), coupling
constants J (torsion angles).
COSY peak patterns of amino acids
Thr
Ala
Leu
Ser, Cys, Asp, Asn
TOCSY patterns of amino acids
TOCSY-NOESY sequential walk in polypeptides
• TOCSY spectrum renders
NH- αH cross peaks (J-
coupled) in fingerprint
region.
• NOESY fingerprint shows
both self & sequential
NOE cross peaks
NHi-αHi (self)
NHi-αH(i-1) (sequential)
From nOe’s
• NH-NH region is the most important followed by the NH-Hα & the
Hα-Hβ regions
Secondary structure elements in peptides
α-helix
• Clockwise spiral, NH’s run upward, CO’s downward
• Stabilised by H-bonds along the chain
• Characterised by short sequential & medium-range 1H-1H distances
β-sheet
• Two peptide strands running alongside each other either in the same or
opposite directions
• Stabilised by H-bonds crossing between the chains
• Characterised by short sequential & long-range backbone distances
β-
turns
• Units of four amino Type I
acids turning back dNN(2,3)
dNN(2,4)
upon themselves dαN(2,4)
• Stabilised by H-bonds
between residues at
the corners Type II
dNN(2,3)
dαN(2,4)
dαN(2,3)
Structure Determination Strategy for nucleic acids
I (H2O) II (H2O)
Assignment of Assignment of non-
imino/amino to establish exchangeable protons
base pairing (NOESY) NOESY imino-
H2/H8/H5/H1’
III (D2O)
Identification of aromatic
Sequential resonance Cytosine/Thymine Identification of sugar
Assignment NOESY H5/H6 spin systems proton spin systems
H6/H8-H1’, H6/H8-H2’H2” COSY/TOCSY COSY/TOCSY
IV (D2O) V
Assignment of 31P resonances & Structure Determination using
confirm/extend H3, H4’, H5’, H5” Torsion Constraints/Distance
assignments. Constraints
(1H,31P ) HETCOR
NOESY-
DNA H2’/2” -> H1’
H2’/2” -> Base
H1’ -> Base
H2’’
ROESY-rotating frame NOESY
• NOE’s measured under spin-locked
conditions
• Suitable for molecules with ωτc~1
where noe’s are absent in NOESY
• Spin exchange between dipolar coupled
spins occurs during the spin-lock period
• Strength of the spin-lock needs to be
optimized to minimise TOCSY & COSY
type artifacts
• Cross peaks are opposite in sign with
diagonal & chemical exchange peaks
Heteronuclear NMR
• For small molecules generally
homonuclear 2D techniques are
sufficient for structure elucidation.
• However, when there is extensive
overlap even in the 2D spectrum it
helps to do heteronuclear
correlation experiments
• It is a technique which helps to
determine which 1H of a molecule is
bonded to which X nucleus in the
molecule. Transfer of magnetization
takes place between nuclei of
different types. The two axis
show the chemical shift of the
respective type of nucleus. If a
transfer has taken place (due to
coupling), a signal appears at the
intersection of the two frequencies,
Heteronuclear correlation
HSQC HMQC HMBC
• one-bond 1H-X • one-bond correlation • two- & three-bond
correlation • Shorter pulse correlation (modified
HMQC)
• Longer pulse sequence & is
program, therefore therefore less • Less sensitive than
sensitive to sensitive to the HMQC
calibration & tuning calibration & tuning • Popular among NMR
errors errors spectroscopists in
• Ideal for • Ideal for small organic labs/pharma
macromolecules, molecules industries
very popular in
protein NMR
C5H8O2
5 2
34
C3
C4
C2
C5
Heteronuclear Corelation in Proteins
• Use of 13C & 15N as NMR-active nuclei
• Facilitate structure determination especially of large
proteins (>100 AA)
• Since both the gyromagnetic ratio as well as the natural
abundance of these is very low as compared to 1H, to
get better sensitivity, these samples are subjected to
isotopic enrichment. i.e. 12C 13
C and 14N 15
N
15
N-1H HSQC of proteins
• The 1H-15N HSQC is the most
important corelation experiment
for the protein NMR spectroscopist
• The number of peaks in its
fingerprint region directly gives the
number of amides in the protein
since there is only one backbone
NH per amino acid.
• It also contains signals from the
NH2 groups of the side chains of
Asn and Gln and of the aromatic
NH protons of Trp and His.
Moral of the story!
Depending on the system you are studying, a
small organic molecule or a complex
biomolecule, a judicious choice from the vast
range of experiments available can be made
which can then make your task of structure
determination faster and simpler.
Thank you
