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Welcome
to the exiting world of NMR…
though a little hard to learn…
remember…
?Why just NMR …
?How a 1D NMR spectrum look like…
?What we can obtain from it…
?and what can we do for a big molecule…
Information from 1D NMR Spectrum
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Chemical Shift ReferenceChemical Shift
J constants
Peak Splitting
Integral Area
Amplitude
Half Height Peak Width
NOE
T1
T2
…
Hydrogen Exchange
生物核磁共振波谱学
NMR in Biological Science
6,2D NMR Spectra
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6.1 INTRODUCTION
? 1971 比利时 Jeener 2D NMR思想
? 1974~76 瑞士 R.R,Ernst 2D NMR理论
? 1979~82 瑞士 K,Wuthrich 2D NMR & 蛋白质
? 1982~ 瑞士 K,Wuthrich
2D NMR +距离 几何 — > 蛋白质空间结构
荷兰 W.F,Van Gunsteren & R,Kaptein
2D NMR + 计算机分子动力学模拟 — > 空间结构
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t1= nDt1 t2
What is 2D NMR?
FID信号的强度和相位与 t2=0时
刻之前核自旋系统的状态有关
S(t1,t2) — 2D FT— > S(w1,w2)
=> Pulse Sequence (Spin Echo Sequence)?
Scheme for 2D pulse sequences
and experiments
Preparation Evolution Acquisition
t1 t2
Mixing
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? The first perturbation of the system (pulse) was
called the preparation of the spin system.
? The variable tE is called the evolution time,t1.
? We have a mixing event,in which information from
one part of the spin system is relayed to other parts.
? Finally,we have an acquisition period (t2) as with all
1D experiments.
以异核 13C－ 1H相关实验为例来说明 2D NMR实验的四个时期：
准备期 (Preparation),发展期 (Evolution)、
混合期 (Magnetization transfer)和 探测期 (detection)。
What is 2D NMR?
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What is 2D NMR?
?Fabian H,Mantsch HH,Schultz CP,PNAS,1999,96,13153–
?Yan YB,Luo XC,Zhou HM,Zhang
RQ,Chinese Sci Bull,2002,47,
389-393
2D IR
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The three pulses in both techniques
are separated by time delays t1 and
t2,and the FID is measured versus
t3,In heterodyned 2D-IR,the three
IR laser beams and the emitted FID
signal beam all travel in different
directions,and thus are spatially
separated from one another,The
electric field of the signal beam
cannot be measured directly,as it is
in NMR,so it is overlapped with a
fourth local-oscillator beam that
heterodynes the signal (not shown),
When t2 = 0,the 2D-IR spectrum is
analogous to a COSY experiment,
otherwise it is similar to NOESY.
? Zanni MT and Hochstrasser RM,Current Opinion in Structural Biology 2001,11:516
Typical pulse sequences used in (a) NMR
spectroscopy and (b) heterodyned 2D-IR
spectroscopy,
Scheme for a 2D NMR spectrum
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Diagonal signals,
results from contributions of the
magnetization that has not been
changed by the mixing sequence
(equal frequency in both dimensions).
Cross signals:
originate from nuclei that exchanged
magnetization during the mixing time
(frequencies of the first and second
nucleus in each dimension,
respectively),They indicate an
interaction of these two nuclei,
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What is 2D NMR?
1） J分辨谱
（ J resolved spectroscopy）
将决定波谱共振位置的两个参数即化
学位移和 J耦合分开,
2）化学位移相关谱
(chemical shift correlation
spectroscopy)
将相互耦合的核、或具有化学交换的
核、或有相互弛豫的核相关联,
3）多量子谱
（ multiple quantum spectroscopy）
检测和应用多量子跃迁,
同核相关谱
异核相关谱
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6.2 同核相关谱
? HOMO2DJ ?
? COSY
? magnitude COSY,phase sensitive COSY,
DQF(MQF)-COSY,RELAY,ECOSY,
? TOCSY
? NOESY
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18090
t1 / 2 t1 / 2
HOMO2DJ
? HOMO2DJ is used to separate chemical shifts and
scalar coupling in different dimensions,
? It involves an evolving spin-echo sequence,in which
the intensity of multiplet lines are modulated by J
coupling in t1.
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d (f2)
J (f1)
0 Hz
2D COSY(Correlated Spectroscopy)
N C
H
C
H
C OH H
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t 1
90x
2t
90x
Cross peaks exist
in the 2-D COSY
spectrum where
there is spin-spin
coupling between
hydrogens,
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H1 H1-H2
H2
H3 H3-H4
H4
http://www.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/2d_nmr.htm
H51
H52
TOCSY
NOESY
HMBC
?
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THR-VAL-LEU
TRY TO ASSIGN IT!
5 min
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Two ways of phase correcting a 2D COSY spectrum,diagonal
peaks in-phase absorptive,crosspeaks antiphase dispersive (left);
or diagonal peaks in-phase dispersive,cross-peaks antiphase
absorptive (right).
The dispersive tails always tend to obscure near-by cross-peaks,
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Disadvantage of COSY
? diagonal peaks are 90 degree out of phase compared
to cross-peaks thus the diagonal peaks are usually
phased to be dispersive so that the cross peaks would
be absorptive.
? Cross peaks only between directly coupled spins are
observed.
? Cross peaks show not only the active coupling but
also passive coupling patterns.
? COSY is sensitive —— A normal COSY can be
obtained in 5 min for a modern NMR spectrometer!
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COSY Variants
? magnitude COSY
? phase sensitive COSY
? MQF-COSY
? E.COSY
? RELAY COSY
2D MQF－ COSY (Multiple-Quantum Filtered COSY)
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aaa
bbb
aba aab baa
abb bba bab p = ? 1
p = ? 2
p = 0
p = ? 3
?The big problem is that we
can only study the
evolution of single-
quantum transitions with
our vector representation.
90 90 90
p = +2
p = + 1
p = 0
p = - 1
p = - 2
?We can add a pulse (with
proper phasing) such that
only double- or higher
quantum transitions selected
by the first and second pulses
will end up in the p = -1 line.
2D DQF－ COSY
(Double-Quantum Filtered COSY)
90?f 90? f 90?x
t1 t2
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MQF-COSY allows for attenuation of the diagonal peaks
and making the peaks along the diagonal have the same
phase as the cross peaks.
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Both cross- and diagonal peaks are derived from a DQC term requiring
the presence of scalar coupling (since it can only be generated from an
antiphase term with the help of another r.f,pulse,2I1yI2z ?? 2I1yI2x),
Therefore,singulet signals – e.g.,solvent signals like H2O! – should be
completely suppressed,even as diagonal signals.
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DQF-COSY Spectra of a protein segment (22 aa )
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Advantage
? MQF-COSY抑制了强峰，包括化合物固有强峰和溶剂峰
? 峰形改善,MQF-COSY获得的是相敏谱，对角峰和交叉
峰都是正负交替的纯吸收峰
? 由于对角线的谱线结构较好，可用来测量 J耦合常数
Disadvantage
?In sensitive thus,time consuming,
相循环的次数是相应 COSY的 4倍 (无梯度场 )
峰的强度为相应 COSY谱的 1/4~1/8
Relay－ COSY
(Double-Quantum Filtered COSY)
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Relayed COSY allows for the observation of cross peaks
between two spins that are not directly coupled but are
both coupled to a third spin.
This sequence is very useful to provide information on
the second neighbor in a complex spectra
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Relayed-COSY spectrum,only the Relayed peaks (in
boxes) show pure (anti-) phase behaviour.
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? singlets fill the 2D spectrum with junk,so
we can do without them,
? we may need to analyze an overlapped
pattern that can be simplified by
removing some of the correlations.
Why do we want to get rid
of certain signals?
90
?
SLxSLx MLEVt1 t2
TOCSY (TOtal Correlation SpectroscopY)
HOHAHA (HOmonuclear HArtmann HAhn)
N C
H
C
H
C OH H
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? TOCSY experiments
correlate all the protons
in the same spin system
? Very useful in complex
molecules with separate
spin systems
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Basics Concepts of TOCSY
?Normally,in a 1D or a COSY,at the Bo external
magnetic field strength,Dd (Hz) >> J (Hz).
? The effects on the energy of the system arising from couplings are
much smaller than those due to chemical shifts,and coherence
transfer between spins is dominated by them,
?If the system is spin-locked,Dd (B1 << Bo) was
removed more or less but not couplings.
? The coupling term dominates the energy of the system,and
coherence transfer occurs due to scalar coupling.
?A certain spin in a coupled system will correlates to all
others in the system.
THNMR
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COSY
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IS4在 DMSO-d6中的 TOCSY谱的区间放大谱
(图中垂直连线显示了各个残基的耦合系统 )
t1 ?m
90° 90° 90°
t2
NOESY
(Nuclear Overhauser Effect Spectroscopy)
N C
H
C
H
C OH H
N C
H
C
H
C OH H
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correlate nuclei
through space
(distance<0.5nm)
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What from NOESY?
?The NOE depends on the strength of the two dipoles
involved,It is proportional to rIS-6.
Hb
Ha
Hc
HbHa Hc
_ =
hab hac
C
? NOE decays very fast as two nuclei apart away from each other,
For protons,this means that we can see things which are at most
5 to 6 ?apart in the molecule (under ideal conditions…).
? Question,Maximum NOE & maximum r?
THNMR
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H1
H2
H3
H4
H51
H52
H1-H52H1-H3
H4-H1
H4-H2
COSY?
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One more thing……
? Consider the sign of the cross-peaks:
Thye will depend on the correlation time tc,in the same way that
the NOE enhancement does,We therefore have positive cross-
peaks for w * tc << 1 and negative cross- peaks for w * tc << 1.
? Consider the range of w * tc values:
the enhancement can be zero,If we express the NOE
enhancement as a function of w * tc we get a sigmoid function,
and we solve it for hI = 0,we get w * tc =1.12,Here,NO NOE
can be observed!!!
? The options are,change tc (kind of hard),w (the magnet is
pretty hard to change…),or trick the system to think that we
are working at a very low field,where w * tc << 1 always.
? The experiment that does this is called ROESY.
Spin-lock
90°
t1 t2
ROESY (ROtating framE SperctroscopY)
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? This technique is based on NOE in the Rotating Frame,
? This pulse sequence is almost identical as TOCSY,To avoid
TOCSY artifacts,the power used to achieve spin-lock is reduced.
? Contrary to NOE that can be positive (for small molecule),
negative (for large molecule) or null (if the correlation time
happen to cancel the NOE),the ROE (NOE in the rotating
frame) is always positive,
? Alternation in sign of the ROE effect allows to distinguish the
"three-spin effect" from true small ROE.
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NOE vs,ROE
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6.3 异核化学位移相关谱
? Heteronuclear 2DJ NMR
? X-detected
? HETeronuclear CORrelation
COLOC,COrrelation of LOng range Coupling
Heteronuclear Proton Relay
1D and 2D INADEQUATE
? H-detected
HMQC,HSQC,HMBC,HMQC-TOCSY,HMQC-NOESY
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HETCOR(HETeronuclear CORrelation)
90
{1H}
1H:
13C:
90 90
t1
The HETCOR sequence reports on which carbon is
attached to what proton and shows them both.
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With JCH Coupling
w13C
w1H
f2
f
1
JCH
w13C
w1H
f2
f
1
With JCH Decoupled
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H-detected (Inverse Mode)
? Low-gamma low-sensitivity X nuclei
when limited amount of material is available,the direct
detection of Carbon-13 can be almost impossible
? High-sensitive protons
?Basic idea:
detection of 1H spectra during t2
- the detection time;
the X chemical shift is detected during t1
- the evolution time.
Good HMQC could be obtained in 2 hr,but direct carbon may
give very noisy spectra after 15 hr.
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HMQC
(Heteronuclear Multiple Quantum Correlation)
The HMQC experiment provides correlation between
protons and their attached heteronuclei through the
heteronuclear scalar coupling,
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HMQC
Each proton-carbon pair has a single crosspeak,
13C nuclei that have diasteriotopic protons have
two correlations (some of these crosspeak pairs
are boxed in the spectrum below).
HMBC
?
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HMBC
(Heteronuclear Multiple Bond Correlation)
This very useful sequence provide information about the skeleton of a
molecule,It is very useful in carbohydrate area as a sequence analysis
tool that provides unique information concerning connectivities across
glycosidic linkages,in the peptide-protein area - specially when
applied to a 15N labeled protein - It is possible with this technique to
get connectivities between the Nitrogen and the CH proton of the
amino acid of the next residue.
The HMBC experiment
detects long range coupling
between proton and carbon
(two or three bonds away)
with great sensitivity.
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COSY
http://www.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/2d_nmr.htm
C2
C5
CO
C1
?
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HMQC
?
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What have we learned?
1） Techniches to simplify the spectra/analysis or to
provide more informations
2） Spectra used to determine the coupling
network/chemical structures
Including:
characterize different spin system
characterize the connective of different spin system
3） Spectra used to determine the space distance and/or
conformation
Including:
NOE (ROE)
dihedrals (through J constants)
2+3 ? NMR solution Structures?
6.4 Solution Structure from NMR
? Ref:
? Wüthrich K,NMR of Proteins
and Nucleic Acids,New York,
John Wiley and Sons,1986
? Wagner G,Nature Structural
Biology,4,841-844 (1997)
? Gaetano T,Montelione et al,
Nature Structural Biology 7,
982 - 985 (2000)
? Wider & wuthrich,Current
opinion in structural biology,9,
594-601 (1999)
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?
?
??
??
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Basic of Protein NMR
where do the peaks of an amino acid show up in the 1H spectrum?
Aromatic
Imines Amides HCb,g,d,...HCa
wa
ter
10 9 8 7 6 5 4 3 2 1 0
? 1H谱的归属
?自旋系统的归属
? Gly,Ala,Thr,Ile,Val,Leu只有一个自旋系统
? Tyr,Phe,His,Trp,Cys,Asp,Asn,Ser只有 AMX
自旋系统
? Glu,Gln和 Met具有 5自旋系统
? Arg,Lys,Pro具有长的自旋系统
?残基内 NH与 aH的连接
?残基序列的归属 (sequence assignment)
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Spin System Assignments
TOCSY,COSY - Used to identify spin systems
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Spin System Assignments
Spin Systems of
Different Amino Acids
Leu
Ala
Asn
Gly
NH
HC
TOCSY
?
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Protein Sequential Assignment
interesidue and sequential NOE,cross-peaks,
which are NOEs among protons of the same
residue and from a residue to protons of the (i + 1)
and (i - 1) residues:
Ni Ci C
O
Ni+1 Ci+1 C
OH2C
R1
H2C
H H HH
R2
daN
dNN
dbN
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Sequential Assignment
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Sequential Assignment
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IS4在三氟乙醇中 500MHz 2DNOESY谱
Sequential Assignment
Tu GZ,et al,
? Chemical Shift
? NOE Distance - NOESY谱中交叉峰的归属
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Structure Constraints
?
?
?
?氢键 (hydrogen bonds)
?二级结构的确定
?Dihedral - 耦合常数的测定
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Molecular Modeling
?As opposed to X-ray,in which we actually ‘see’ the electron
density from atoms in the molecule and can be considered as a
‘direct’ method,with NMR we only get indirect information on
some atoms of the molecule (mainly 1Hs…).
? Therefore,we will have to rely on some form of theoretical model
to represent the structure of the peptide,Usually this means a
computer generated molecular model.
?A molecular model can have different degrees of complexity:
? ab initio
? Semiempirical
? Molecular mechanics (MM)
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Structure Optimization
? DG (distance geometry ).
? MD (Molecular Dynamics)
? SA (Simulated Annealing)
?
?
?
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Structure optimization
?Etotal ?Etotal
> 0 Etotal,< 0 Etotal?
xyz ?xyz
? We have energy barriers
and valleys (minima).
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Presentation of results
the floppiness of certain
regions is an indication of
the lack of NOE constrains,
which reflects the real
flexibility of the molecule in
solution.
? So far we obtained a collection of low energy structures
which have no big violations of the constraints or are in
agreement with the NMR data.
? A set of structures superimposed along the most fixed parts
of the molecule.
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seminar
and what next …
A-Z Applications…
—— based on fundamental NMR assignments and may be
structure calculation
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Thank you!
and what next class…
Seminar
if possible...
Brief intro to isotope labelling
Brief intro to multi-D NMR
Brief intro to new NMR techniques
and anything you interested…
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The Spin-Echo Sequence
90x— TE— 180x— TE— AQ
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The Spin-Echo Sequence
Different Chemical Shift
Refocus
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The Spin-Echo Sequence
J Coupling
—— AX System
Signal was
modulated by
cos(2pJ·TE)
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The Spin-Echo Sequence
90x— TE— 180x— TE— AQ
A2-C-C-B
2-D J-resolved NMR experiment
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Couplings and dihedrals
3JNa f
3Jab c N
N
O
H
H
Ha
Hb
Hb
AA
f
c
y
w
3JNa,which reports on the conformation of the peptide
backbone,and 3Jab which is related to the side chain
conformation
?
Case 1 2 3
Conformation g2g3 g2t3 t2g3
c1
3J
ab2(Hz)
3J
ab3(Hz)
NOE(a,b2)
NOE(a,b3)
NOE(NH,b2)
NOE(NH,b3)
60°
3.4
3.4
Strong
Strong
Weak
Strong
180°
3.4
12.9
Strong
Weak
Strong
Strong
-60°
12.9
3.4
Weak
Strong
Strong
Weak
Hb3
Hb2 R1
Ca
C O
Ha
H N
Hb2
R1 Hb 3
Ca
C O
Ha
HN
R1
Hb3 Hb 2
Ca
C O
Ha
H N
二面角 c 1 的 N M R 确定THNMR
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Hydrogen Bonds
4.0
4.0
(Has)
4.0
8.0 (NHs) 7.0
t = 0 - No D2O
Add D2O
t = t1
t = t2
重氢交换 13小时后的 2D TOCSY谱指纹区（右下区）的放大图
Hydrogen Bonds
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Secondary Structure
i+4
i+3
i+2
i
i-1
i+1
dab(i,i+3)
daN(i,i+3)
dNN(i,i+3)
daN(i,i+4)
a-helices
C
N
C
N
N
C
N
C
da(i)N(j)
b-sheets
regular secondary structure will have:
(1) regular NOE patterns
(2) regular Hydrogen Bonds
(3) regular Chemical Shift Index
?
不同的二级结构中相邻或中程质子－质子间的距离（单位,nm） 和邻位偶合常数（ Hz）
a-helix 310-helix b-antiparallel b-parallel
f=－ 57? f=－ 60? f=－ 139? f=－ 119?
y=－ 47? y=－ 30? y=＋ 135? y=＋ 113?
daN(i,i) 0.27 0.27 0.28 0.28
daN(i,i+1) 0.35 0.34 0.22 0.22
daN(i,i+2) 0.44 0.38
daN(i,i+3) 0.34 0.33
daN(i,i+4) 0.42 (>0.45)
dNN(i,i+1) 0.28 0.26 0.43 0.42
dNN(i,i+2) 0.42 0.41
dNN(i,i+3) (0.48) (0.52)
dab(i,i+3) 0.25-0.44 0.31-0.51
3JHNa 4 4 10 10
3J(HaiNi+1) -6 -6 -2 -2
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规则的平行与反平行 b－折叠片中的
特征距离 （ × 0.1 nm）
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四种最常见转角的结构及其特征参数
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CSI (Chemical Shift Index) 0f
Secondary Structure
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Welcome
to the exiting world of NMR…
though a little hard to learn…
remember…
?Why just NMR …
?How a 1D NMR spectrum look like…
?What we can obtain from it…
?and what can we do for a big molecule…
Information from 1D NMR Spectrum
THNMR
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Chemical Shift ReferenceChemical Shift
J constants
Peak Splitting
Integral Area
Amplitude
Half Height Peak Width
NOE
T1
T2
…
Hydrogen Exchange
生物核磁共振波谱学
NMR in Biological Science
6,2D NMR Spectra
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6.1 INTRODUCTION
? 1971 比利时 Jeener 2D NMR思想
? 1974~76 瑞士 R.R,Ernst 2D NMR理论
? 1979~82 瑞士 K,Wuthrich 2D NMR & 蛋白质
? 1982~ 瑞士 K,Wuthrich
2D NMR +距离 几何 — > 蛋白质空间结构
荷兰 W.F,Van Gunsteren & R,Kaptein
2D NMR + 计算机分子动力学模拟 — > 空间结构
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t1= nDt1 t2
What is 2D NMR?
FID信号的强度和相位与 t2=0时
刻之前核自旋系统的状态有关
S(t1,t2) — 2D FT— > S(w1,w2)
=> Pulse Sequence (Spin Echo Sequence)?
Scheme for 2D pulse sequences
and experiments
Preparation Evolution Acquisition
t1 t2
Mixing
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? The first perturbation of the system (pulse) was
called the preparation of the spin system.
? The variable tE is called the evolution time,t1.
? We have a mixing event,in which information from
one part of the spin system is relayed to other parts.
? Finally,we have an acquisition period (t2) as with all
1D experiments.
以异核 13C－ 1H相关实验为例来说明 2D NMR实验的四个时期：
准备期 (Preparation),发展期 (Evolution)、
混合期 (Magnetization transfer)和 探测期 (detection)。
What is 2D NMR?
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What is 2D NMR?
?Fabian H,Mantsch HH,Schultz CP,PNAS,1999,96,13153–
?Yan YB,Luo XC,Zhou HM,Zhang
RQ,Chinese Sci Bull,2002,47,
389-393
2D IR
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The three pulses in both techniques
are separated by time delays t1 and
t2,and the FID is measured versus
t3,In heterodyned 2D-IR,the three
IR laser beams and the emitted FID
signal beam all travel in different
directions,and thus are spatially
separated from one another,The
electric field of the signal beam
cannot be measured directly,as it is
in NMR,so it is overlapped with a
fourth local-oscillator beam that
heterodynes the signal (not shown),
When t2 = 0,the 2D-IR spectrum is
analogous to a COSY experiment,
otherwise it is similar to NOESY.
? Zanni MT and Hochstrasser RM,Current Opinion in Structural Biology 2001,11:516
Typical pulse sequences used in (a) NMR
spectroscopy and (b) heterodyned 2D-IR
spectroscopy,
Scheme for a 2D NMR spectrum
THNMR
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Diagonal signals,
results from contributions of the
magnetization that has not been
changed by the mixing sequence
(equal frequency in both dimensions).
Cross signals:
originate from nuclei that exchanged
magnetization during the mixing time
(frequencies of the first and second
nucleus in each dimension,
respectively),They indicate an
interaction of these two nuclei,
THNMR
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What is 2D NMR?
1） J分辨谱
（ J resolved spectroscopy）
将决定波谱共振位置的两个参数即化
学位移和 J耦合分开,
2）化学位移相关谱
(chemical shift correlation
spectroscopy)
将相互耦合的核、或具有化学交换的
核、或有相互弛豫的核相关联,
3）多量子谱
（ multiple quantum spectroscopy）
检测和应用多量子跃迁,
同核相关谱
异核相关谱
THNMR
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6.2 同核相关谱
? HOMO2DJ ?
? COSY
? magnitude COSY,phase sensitive COSY,
DQF(MQF)-COSY,RELAY,ECOSY,
? TOCSY
? NOESY
THNMR
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18090
t1 / 2 t1 / 2
HOMO2DJ
? HOMO2DJ is used to separate chemical shifts and
scalar coupling in different dimensions,
? It involves an evolving spin-echo sequence,in which
the intensity of multiplet lines are modulated by J
coupling in t1.
THNMR
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d (f2)
J (f1)
0 Hz
2D COSY(Correlated Spectroscopy)
N C
H
C
H
C OH H
THNMR
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t 1
90x
2t
90x
Cross peaks exist
in the 2-D COSY
spectrum where
there is spin-spin
coupling between
hydrogens,
THNMR
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THNMR
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H1 H1-H2
H2
H3 H3-H4
H4
http://www.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/2d_nmr.htm
H51
H52
TOCSY
NOESY
HMBC
?
THNMR
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THR-VAL-LEU
TRY TO ASSIGN IT!
5 min
THNMR
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Two ways of phase correcting a 2D COSY spectrum,diagonal
peaks in-phase absorptive,crosspeaks antiphase dispersive (left);
or diagonal peaks in-phase dispersive,cross-peaks antiphase
absorptive (right).
The dispersive tails always tend to obscure near-by cross-peaks,
THNMR
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Disadvantage of COSY
? diagonal peaks are 90 degree out of phase compared
to cross-peaks thus the diagonal peaks are usually
phased to be dispersive so that the cross peaks would
be absorptive.
? Cross peaks only between directly coupled spins are
observed.
? Cross peaks show not only the active coupling but
also passive coupling patterns.
? COSY is sensitive —— A normal COSY can be
obtained in 5 min for a modern NMR spectrometer!
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COSY Variants
? magnitude COSY
? phase sensitive COSY
? MQF-COSY
? E.COSY
? RELAY COSY
2D MQF－ COSY (Multiple-Quantum Filtered COSY)
THNMR
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aaa
bbb
aba aab baa
abb bba bab p = ? 1
p = ? 2
p = 0
p = ? 3
?The big problem is that we
can only study the
evolution of single-
quantum transitions with
our vector representation.
90 90 90
p = +2
p = + 1
p = 0
p = - 1
p = - 2
?We can add a pulse (with
proper phasing) such that
only double- or higher
quantum transitions selected
by the first and second pulses
will end up in the p = -1 line.
2D DQF－ COSY
(Double-Quantum Filtered COSY)
90?f 90? f 90?x
t1 t2
THNMR
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MQF-COSY allows for attenuation of the diagonal peaks
and making the peaks along the diagonal have the same
phase as the cross peaks.
THNMR
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Both cross- and diagonal peaks are derived from a DQC term requiring
the presence of scalar coupling (since it can only be generated from an
antiphase term with the help of another r.f,pulse,2I1yI2z ?? 2I1yI2x),
Therefore,singulet signals – e.g.,solvent signals like H2O! – should be
completely suppressed,even as diagonal signals.
THNMR
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DQF-COSY Spectra of a protein segment (22 aa )
THNMR
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Advantage
? MQF-COSY抑制了强峰，包括化合物固有强峰和溶剂峰
? 峰形改善,MQF-COSY获得的是相敏谱，对角峰和交叉
峰都是正负交替的纯吸收峰
? 由于对角线的谱线结构较好，可用来测量 J耦合常数
Disadvantage
?In sensitive thus,time consuming,
相循环的次数是相应 COSY的 4倍 (无梯度场 )
峰的强度为相应 COSY谱的 1/4~1/8
Relay－ COSY
(Double-Quantum Filtered COSY)
THNMR
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Relayed COSY allows for the observation of cross peaks
between two spins that are not directly coupled but are
both coupled to a third spin.
This sequence is very useful to provide information on
the second neighbor in a complex spectra
THNMR
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Relayed-COSY spectrum,only the Relayed peaks (in
boxes) show pure (anti-) phase behaviour.
THNMR
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? singlets fill the 2D spectrum with junk,so
we can do without them,
? we may need to analyze an overlapped
pattern that can be simplified by
removing some of the correlations.
Why do we want to get rid
of certain signals?
90
?
SLxSLx MLEVt1 t2
TOCSY (TOtal Correlation SpectroscopY)
HOHAHA (HOmonuclear HArtmann HAhn)
N C
H
C
H
C OH H
THNMR
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? TOCSY experiments
correlate all the protons
in the same spin system
? Very useful in complex
molecules with separate
spin systems
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Basics Concepts of TOCSY
?Normally,in a 1D or a COSY,at the Bo external
magnetic field strength,Dd (Hz) >> J (Hz).
? The effects on the energy of the system arising from couplings are
much smaller than those due to chemical shifts,and coherence
transfer between spins is dominated by them,
?If the system is spin-locked,Dd (B1 << Bo) was
removed more or less but not couplings.
? The coupling term dominates the energy of the system,and
coherence transfer occurs due to scalar coupling.
?A certain spin in a coupled system will correlates to all
others in the system.
THNMR
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COSY
THNMR
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IS4在 DMSO-d6中的 TOCSY谱的区间放大谱
(图中垂直连线显示了各个残基的耦合系统 )
t1 ?m
90° 90° 90°
t2
NOESY
(Nuclear Overhauser Effect Spectroscopy)
N C
H
C
H
C OH H
N C
H
C
H
C OH H
THNMR
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correlate nuclei
through space
(distance<0.5nm)
THNMR
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What from NOESY?
?The NOE depends on the strength of the two dipoles
involved,It is proportional to rIS-6.
Hb
Ha
Hc
HbHa Hc
_ =
hab hac
C
? NOE decays very fast as two nuclei apart away from each other,
For protons,this means that we can see things which are at most
5 to 6 ?apart in the molecule (under ideal conditions…).
? Question,Maximum NOE & maximum r?
THNMR
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H1
H2
H3
H4
H51
H52
H1-H52H1-H3
H4-H1
H4-H2
COSY?
THNMR
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One more thing……
? Consider the sign of the cross-peaks:
Thye will depend on the correlation time tc,in the same way that
the NOE enhancement does,We therefore have positive cross-
peaks for w * tc << 1 and negative cross- peaks for w * tc << 1.
? Consider the range of w * tc values:
the enhancement can be zero,If we express the NOE
enhancement as a function of w * tc we get a sigmoid function,
and we solve it for hI = 0,we get w * tc =1.12,Here,NO NOE
can be observed!!!
? The options are,change tc (kind of hard),w (the magnet is
pretty hard to change…),or trick the system to think that we
are working at a very low field,where w * tc << 1 always.
? The experiment that does this is called ROESY.
Spin-lock
90°
t1 t2
ROESY (ROtating framE SperctroscopY)
THNMR
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? This technique is based on NOE in the Rotating Frame,
? This pulse sequence is almost identical as TOCSY,To avoid
TOCSY artifacts,the power used to achieve spin-lock is reduced.
? Contrary to NOE that can be positive (for small molecule),
negative (for large molecule) or null (if the correlation time
happen to cancel the NOE),the ROE (NOE in the rotating
frame) is always positive,
? Alternation in sign of the ROE effect allows to distinguish the
"three-spin effect" from true small ROE.
THNMR
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NOE vs,ROE
THNMR
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6.3 异核化学位移相关谱
? Heteronuclear 2DJ NMR
? X-detected
? HETeronuclear CORrelation
COLOC,COrrelation of LOng range Coupling
Heteronuclear Proton Relay
1D and 2D INADEQUATE
? H-detected
HMQC,HSQC,HMBC,HMQC-TOCSY,HMQC-NOESY
THNMR
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HETCOR(HETeronuclear CORrelation)
90
{1H}
1H:
13C:
90 90
t1
The HETCOR sequence reports on which carbon is
attached to what proton and shows them both.
THNMR
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With JCH Coupling
w13C
w1H
f2
f
1
JCH
w13C
w1H
f2
f
1
With JCH Decoupled
THNMR
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H-detected (Inverse Mode)
? Low-gamma low-sensitivity X nuclei
when limited amount of material is available,the direct
detection of Carbon-13 can be almost impossible
? High-sensitive protons
?Basic idea:
detection of 1H spectra during t2
- the detection time;
the X chemical shift is detected during t1
- the evolution time.
Good HMQC could be obtained in 2 hr,but direct carbon may
give very noisy spectra after 15 hr.
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HMQC
(Heteronuclear Multiple Quantum Correlation)
The HMQC experiment provides correlation between
protons and their attached heteronuclei through the
heteronuclear scalar coupling,
THNMR
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HMQC
Each proton-carbon pair has a single crosspeak,
13C nuclei that have diasteriotopic protons have
two correlations (some of these crosspeak pairs
are boxed in the spectrum below).
HMBC
?
THNMR
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HMBC
(Heteronuclear Multiple Bond Correlation)
This very useful sequence provide information about the skeleton of a
molecule,It is very useful in carbohydrate area as a sequence analysis
tool that provides unique information concerning connectivities across
glycosidic linkages,in the peptide-protein area - specially when
applied to a 15N labeled protein - It is possible with this technique to
get connectivities between the Nitrogen and the CH proton of the
amino acid of the next residue.
The HMBC experiment
detects long range coupling
between proton and carbon
(two or three bonds away)
with great sensitivity.
THNMR
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COSY
http://www.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/2d_nmr.htm
C2
C5
CO
C1
?
THNMR
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HMQC
?
THNMR
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What have we learned?
1） Techniches to simplify the spectra/analysis or to
provide more informations
2） Spectra used to determine the coupling
network/chemical structures
Including:
characterize different spin system
characterize the connective of different spin system
3） Spectra used to determine the space distance and/or
conformation
Including:
NOE (ROE)
dihedrals (through J constants)
2+3 ? NMR solution Structures?
6.4 Solution Structure from NMR
? Ref:
? Wüthrich K,NMR of Proteins
and Nucleic Acids,New York,
John Wiley and Sons,1986
? Wagner G,Nature Structural
Biology,4,841-844 (1997)
? Gaetano T,Montelione et al,
Nature Structural Biology 7,
982 - 985 (2000)
? Wider & wuthrich,Current
opinion in structural biology,9,
594-601 (1999)
THNMR
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?
?
??
??
THNMR
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Basic of Protein NMR
where do the peaks of an amino acid show up in the 1H spectrum?
Aromatic
Imines Amides HCb,g,d,...HCa
wa
ter
10 9 8 7 6 5 4 3 2 1 0
? 1H谱的归属
?自旋系统的归属
? Gly,Ala,Thr,Ile,Val,Leu只有一个自旋系统
? Tyr,Phe,His,Trp,Cys,Asp,Asn,Ser只有 AMX
自旋系统
? Glu,Gln和 Met具有 5自旋系统
? Arg,Lys,Pro具有长的自旋系统
?残基内 NH与 aH的连接
?残基序列的归属 (sequence assignment)
THNMR
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Spin System Assignments
TOCSY,COSY - Used to identify spin systems
THNMR
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Spin System Assignments
Spin Systems of
Different Amino Acids
Leu
Ala
Asn
Gly
NH
HC
TOCSY
?
THNMR
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Protein Sequential Assignment
interesidue and sequential NOE,cross-peaks,
which are NOEs among protons of the same
residue and from a residue to protons of the (i + 1)
and (i - 1) residues:
Ni Ci C
O
Ni+1 Ci+1 C
OH2C
R1
H2C
H H HH
R2
daN
dNN
dbN
THNMR
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Sequential Assignment
THNMR
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Sequential Assignment
THNMR
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IS4在三氟乙醇中 500MHz 2DNOESY谱
Sequential Assignment
Tu GZ,et al,
? Chemical Shift
? NOE Distance - NOESY谱中交叉峰的归属
THNMR
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Structure Constraints
?
?
?
?氢键 (hydrogen bonds)
?二级结构的确定
?Dihedral - 耦合常数的测定
THNMR
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Molecular Modeling
?As opposed to X-ray,in which we actually ‘see’ the electron
density from atoms in the molecule and can be considered as a
‘direct’ method,with NMR we only get indirect information on
some atoms of the molecule (mainly 1Hs…).
? Therefore,we will have to rely on some form of theoretical model
to represent the structure of the peptide,Usually this means a
computer generated molecular model.
?A molecular model can have different degrees of complexity:
? ab initio
? Semiempirical
? Molecular mechanics (MM)
THNMR
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Structure Optimization
? DG (distance geometry ).
? MD (Molecular Dynamics)
? SA (Simulated Annealing)
?
?
?
THNMR
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Structure optimization
?Etotal ?Etotal
> 0 Etotal,< 0 Etotal?
xyz ?xyz
? We have energy barriers
and valleys (minima).
THNMR
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Presentation of results
the floppiness of certain
regions is an indication of
the lack of NOE constrains,
which reflects the real
flexibility of the molecule in
solution.
? So far we obtained a collection of low energy structures
which have no big violations of the constraints or are in
agreement with the NMR data.
? A set of structures superimposed along the most fixed parts
of the molecule.
THNMR
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THNMR
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seminar
and what next …
A-Z Applications…
—— based on fundamental NMR assignments and may be
structure calculation
THNMR
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Thank you!
and what next class…
Seminar
if possible...
Brief intro to isotope labelling
Brief intro to multi-D NMR
Brief intro to new NMR techniques
and anything you interested…
THNMR
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The Spin-Echo Sequence
90x— TE— 180x— TE— AQ
THNMR
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The Spin-Echo Sequence
Different Chemical Shift
Refocus
THNMR
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The Spin-Echo Sequence
J Coupling
—— AX System
Signal was
modulated by
cos(2pJ·TE)
THNMR
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The Spin-Echo Sequence
90x— TE— 180x— TE— AQ
A2-C-C-B
2-D J-resolved NMR experiment
THNMR
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THNMR
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Couplings and dihedrals
3JNa f
3Jab c N
N
O
H
H
Ha
Hb
Hb
AA
f
c
y
w
3JNa,which reports on the conformation of the peptide
backbone,and 3Jab which is related to the side chain
conformation
?
Case 1 2 3
Conformation g2g3 g2t3 t2g3
c1
3J
ab2(Hz)
3J
ab3(Hz)
NOE(a,b2)
NOE(a,b3)
NOE(NH,b2)
NOE(NH,b3)
60°
3.4
3.4
Strong
Strong
Weak
Strong
180°
3.4
12.9
Strong
Weak
Strong
Strong
-60°
12.9
3.4
Weak
Strong
Strong
Weak
Hb3
Hb2 R1
Ca
C O
Ha
H N
Hb2
R1 Hb 3
Ca
C O
Ha
HN
R1
Hb3 Hb 2
Ca
C O
Ha
H N
二面角 c 1 的 N M R 确定THNMR
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THNMR
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Hydrogen Bonds
4.0
4.0
(Has)
4.0
8.0 (NHs) 7.0
t = 0 - No D2O
Add D2O
t = t1
t = t2
重氢交换 13小时后的 2D TOCSY谱指纹区（右下区）的放大图
Hydrogen Bonds
THNMR
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Secondary Structure
i+4
i+3
i+2
i
i-1
i+1
dab(i,i+3)
daN(i,i+3)
dNN(i,i+3)
daN(i,i+4)
a-helices
C
N
C
N
N
C
N
C
da(i)N(j)
b-sheets
regular secondary structure will have:
(1) regular NOE patterns
(2) regular Hydrogen Bonds
(3) regular Chemical Shift Index
?
不同的二级结构中相邻或中程质子－质子间的距离（单位,nm） 和邻位偶合常数（ Hz）
a-helix 310-helix b-antiparallel b-parallel
f=－ 57? f=－ 60? f=－ 139? f=－ 119?
y=－ 47? y=－ 30? y=＋ 135? y=＋ 113?
daN(i,i) 0.27 0.27 0.28 0.28
daN(i,i+1) 0.35 0.34 0.22 0.22
daN(i,i+2) 0.44 0.38
daN(i,i+3) 0.34 0.33
daN(i,i+4) 0.42 (>0.45)
dNN(i,i+1) 0.28 0.26 0.43 0.42
dNN(i,i+2) 0.42 0.41
dNN(i,i+3) (0.48) (0.52)
dab(i,i+3) 0.25-0.44 0.31-0.51
3JHNa 4 4 10 10
3J(HaiNi+1) -6 -6 -2 -2
THNMR
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规则的平行与反平行 b－折叠片中的
特征距离 （ × 0.1 nm）
THNMR
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四种最常见转角的结构及其特征参数
THNMR
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THNMR
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CSI (Chemical Shift Index) 0f
Secondary Structure
