2d nmr: assignments & applicationsiupab/2d-learning-ws2009-new.pdf · applications in...

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2D NMR: 2D NMR: Applications Applications IUPAB sponsored IUPAB sponsored Workshop on NMR & its Workshop on NMR & its Applications in Biological Systems” Applications in Biological Systems” November 23-30, 2009 November 23-30, 2009 Mamata Joshi Mamata Joshi TIFR, Mumbai TIFR, Mumbai

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Page 1: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

2D NMR: 2D NMR: ApplicationsApplications

““IUPAB sponsoredIUPAB sponsored Workshop on NMR & its Workshop on NMR & its Applications in Biological Systems”Applications in Biological Systems”

November 23-30, 2009 November 23-30, 2009

Mamata JoshiMamata JoshiTIFR, MumbaiTIFR, Mumbai

Page 2: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

What is 2D NMR?What is 2D NMR?

When and Why?When and Why?

How to apply?How to apply?

Page 3: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

WHAT?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

Page 4: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

2D spectrum – actual view2D spectrum – actual view

Page 5: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

1H chemical shift (ppm)

1 H c

hem

ical

shi

ft (p

pm) diagonal peak (F1=F2)

crosspeak: correlation of twodifferent resonancesby short interatomicdistance or through-bondconnection

HA HB

HA HB

Basic features of 2D spectraBasic features of 2D spectra

Page 6: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

WHEN?WHEN?

Overlap of signals in the 1D spectrum

No overlap, easy to assign

Page 7: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Scalar coupling (J) based• COSY -COrelation SpectroscopY• TOCSY- TOtal Corelation

SpectroscopY• MQF- Multiple Quantum Filter

spectroscopy

NOE based• NOESY- Nuclear Overhauser

Effect SpectroscopY• ROESY- ROtating framE

SpectroscopY

WHY?WHY?What information are you seeking?• 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

Corelation?

Page 8: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

2D-NMR sequences contain two types2D-NMR sequences contain two typesof time periods:of time periods:

evolution time and mixing timeevolution time and mixing time

evolution time:a second frequencydimension is created byindirect detection

mixing time:magnetisation istransferred from spin tospin via interactionsbetween spins

Page 9: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

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+ CCos2θA, B & C are empirical

constants

Page 10: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

• 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

COSYCOSY

Page 11: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

CC33HH88OO

CH3CH2CH2 OH

CH3 – CH2 – CH2 – OH

Page 12: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

CC33HH88O: COSYO: COSY

CH3 – CH2 – CH2 – OH CH3

CH2

CH2 OH

Page 13: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

• 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

DQF COSYDQF COSY

Page 14: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

COSY Vs DQFCOSYCOSY Vs DQFCOSY

Page 15: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

• 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.

TOCSYTOCSY

Page 16: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Other COSY-type experimentsOther COSY-type experiments• COSYLR

Long range coupling networks probed

• HOMO2DJ-Resolverenders J directly on the F1-axis of the 2D plane with δ on the F2-axis

• ECOSYsimplifies complex splitting patterns by retaining only direct couplings & eliminating signals due to passive coupling

J-Resolve

Page 17: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

NOE-based experimentsNOE-based experimentsNOESY

• 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

Page 18: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Mixing time τm

• 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

Page 19: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

NOESY applicationsNOESY applicationsWhich isomer is responsible for this spectrum?Which isomer is responsible for this spectrum?

Page 20: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

H3H4

H5

OMe

NOESYNOESY

Page 21: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

• 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 Dynein light chain protein (DLC8) (DLC8) 89 residues, MW~10.3kD89 residues, MW~10.3kD

NMR of BiomoleculesNMR of Biomolecules

Page 22: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Structure Determination Strategy for Structure Determination Strategy for PolypeptidesPolypeptides

• 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).

Page 23: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Leu

COSY peak patterns of amino acids COSY peak patterns of amino acids ThrAla

Ser, Cys, Asp, Asn

Page 24: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

TOCSY patterns of amino acidsTOCSY patterns of amino acids

Page 25: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

TOCSY-NOESY sequential walk in polypeptidesTOCSY-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 peaksNHi-αHi (self)NHi-αH(i-1) (sequential)

Page 26: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

From nOe’sFrom nOe’s• NH-NH region is the most important followed by the NH-Hα & the

Hα-Hβ regions

Page 27: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Secondary structure elements in peptidesSecondary structure elements in peptidesαα-helix-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

Page 28: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

ββ-sheet-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

Page 29: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

ββ--turnsturns

Type IdNN(2,3)

dNN(2,4)

dαN(2,4)

Type IIdNN(2,3)

dαN(2,4)

dαN(2,3)

• Units of four amino acids turning back upon themselves

• Stabilised by H-bonds between residues at the corners

Page 30: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Structure Determination Strategy for nucleic acidsStructure Determination Strategy for nucleic acids

Assignment of imino/amino to establish base pairing (NOESY)

Assignment of non-exchangeable protons NOESY imino-H2/H8/H5/H1’

Identification of sugar proton spin systemsCOSY/TOCSY

Identification of aromatic Cytosine/ThymineH5/H6 spin systemsCOSY/TOCSY

Assignment of 31P resonances & confirm/extend H3, H4’, H5’, H5” assignments.(1H,31P ) HETCOR

Structure Determination using Torsion Constraints/Distance Constraints

Sequential resonanceAssignment NOESYH6/H8-H1’, H6/H8-H2’H2”

I (H2O) II (H2O)

III (D2O)

IV (D2O) V

Page 31: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

NOESY-NOESY-DNADNA

H1’H1’ -> Base -> Base

H2’/2” H2’/2” -> Base-> Base

H2’/2” H2’/2” -> -> H1’ H1’

H2’’

Page 32: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

ROESY-rotating frame NOESYROESY-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

Page 33: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Heteronuclear NMRHeteronuclear 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 magnetizationtakes place between nuclei ofdifferent types. The two axisshow the chemical shift of therespective type of nucleus. If atransfer has taken place (due to coupling), a signal appears at theintersection of the two frequencies,

Page 34: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Heteronuclear correlationHeteronuclear correlation

HSQC• one-bond 1H-X

correlation• Longer pulse

program, therefore sensitive to calibration & tuning errors

• Ideal for macromolecules, very popular in protein NMR

HMQC• one-bond correlation • Shorter pulse

sequence & is therefore less sensitive to calibration & tuning errors

• Ideal for small molecules

HMBC• two- & three-bond

correlation (modified HMQC)

• Less sensitive than the HMQC

• Popular among NMR spectroscopists in organic labs/pharma industries

Page 35: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

CC55HH88OO22

234

5

C5

C2

C3C4

Page 36: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Heteronuclear Corelation in Proteins 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 13C and 14N 15N

Page 37: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

1515N-N-11H HSQC of proteinsH 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.

Page 38: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Moral of the story!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.

Page 39: 2D NMR: Assignments & Applicationsiupab/2d-learning-WS2009-new.pdf · Applications in Biological Systems ... • Ideal for assignment of spins in biomolecules (polypeptides/nucleotides)

Thank youThank you