Transcript NMR theory and experiments
NMR theory and experiments
NMR theory and experiments
Analytical Chemistry TIGP, Academia Sinica Instructor: Der-Lii M. Tzou Hour: 9:00~12:00 am June 6, 2007 (02) 2789-8524 email:[email protected]
Place: A508, IC •
NMR basics and principle
(a) Rotation spectroscopy (b) Larmor frequency (c) Resonance, Fourier transfer •
Applications of NMR to Biological Systems
(a) 1D NMR and chemical shifts (b) J-coupling (c) T1 and T2 relaxation (d) NOE and 2D NOE spectroscopy (d) 2D TOCSY spectroscopy (e) 2D COSY spectroscopy (f) 1 H, 13 C, 15 N NMR spectroscopy and high resolution multi-dimensional NMR (g) Other nuclear spin interactions
Without External Magnetic Field
B o
B o
>>
M o
m i
= M o
0
Number of spin states (2I
＋
1) :
A nucleus with spin I can have 2I
＋
1 spin
states. Each of these states has its own spin
quantum number m ( m
＝－
I,
－
I
＋
1,…, I
－
1, I ). For nuclei with I
＝
1/2, only two states are possible : m
＝＋
1/2 and m
＝－
1/2.
E
0
Nuclear Zeeman effect :
ΔE m=
－
1/2 E
＝－
mB 0 (γh/2π) B 0 : magnetic field strength h : Planck’s constant m : spin quantum number
γ
: magnetogyric ratio m=
＋
1/2 B 0
Larmor Frequency
FI D puls e Data acquisition (detection) dela y tim e
Z M (
∥
B 0 ) Z X B 1 Y X 2 M Y 1 M
3D-FID (Free Induction Decay)
Fourier Transform
Examples :
8
CH 3 H H H
6
CH 3
4 Chemical shift
(ppm) 2
CH 4 (CH 3 ) 4 Si CH 3 Li
0 -2 downfield high frequency Increased shielding Increased deshielding upfield low frequency
1H-1H spin-spin J-coupling observed by NMR
The effective magnetic field is also affected by the orientation of neighboring nuclei. This effect is known as spin-spin coupling which can cause splitting of the signal for each type of nucleus into two or more lines.
Returning to the example of ethylbenzene, the methyl (CH3) group has a coupling pattern in the form of A3X2 which to a first order approximation looks like an AX2 multiplet. Likewise, the methylene (CH2) group has the form A2X3 that is equivalent to AX3. The first order approximation works because the groups are widely separated in the spectrum. The aromatic signals are close together and display second order effects. The
ortho
signal is a doublet AX while the
meta
and
para
signals are triplets
Isolated spins (Zeeman effect, Larmor Frequency) Electron cloud (chemical shift) Scalar coupling (J-coupling through chemical bonds) Dipolar interaction (NOE through space) * Spin dynamics (T1, T2… relaxations)
Longitudinal relaxation or spin-relaxation relaxation T 1
Longitudinal relaxation (
T 1
) is the mechanism by which an excited magnetization vector returns to equilibrium (conventionally shown along the
z
axis).
Inversion recovery curve for the methyl protons of ethylbenzene (0.1%) in CDCl 3 at 400 Mhz
The inversion recovery (
T
1) pulse sequence yields a signal of intensity
I
0 1 2 exp
T
1
Transverse relaxation T 2 , spin-spin relaxation
Transverse relaxation (
T
2) is the mechanism by which the excited magnetization vector (conventionally shown in the x-y plane) decays. This is always at least slightly faster than longitudinal relaxation.
Spin-echo experiment for proton spectrum of ethylbenzene (0.1%) in CDCl 3 at 400 MHz, the residual water peak on the left relaxes faster than the methyl on the right.
Exponential decay curve for the methyl protons of ethylbenzene (0.1%) in CDCl3 at 400 MHz
One Dimensional NMR Spectroscopy The 1D experiment Each 1D NMR experiment consists of two sections: preparation and detection. During preparation the spin system is set to a defined state. During detection the resulting signal is recorded. In the simplest case the preparation is a 90 degree pulse (in our example applied along the x axis) which rotates the equilibrium magnetization M z onto the y axis (M y ). After this pulse each spin precesses with its own Larmor frequency around the z axis and induces a signal in the receiver coil. The signal decays due to T 2 relaxation and is therefore called free induction decay (FID). Usually, the experiment is repeated several times and the data are summed up to increase the signal to noise ratio. After summation the data are fourier transformed to yield the final 1D spectrum.
Anatomy of a 2D experiment 90 x t 1 90 y t 2 FID Preparation Evolution Mixing Time Detection The construction of a 2D experiment is simple: in addition to preparation and detection which are already known from 1D experiments the 2D experiment has indirect evolution time t1 and a mixing sequence. The scheme can be viewed as : Do something with the nuclei (preparation), let them precess freely (evolution), do something else (mixing), and detect the result (detection, of course)
t Anatomy of a 3D experiment 1 m t 2 MLEV t 3 FID Preparation Evolution Mixing Time Evolution Mixing Time Detection A three dimensional NMR experiment (see picture above) can easily be constructed from a two-dimensional one by inserting an additional indirect evolution time and a second mixing period between the first mixing period and the direct data acqusition. Each of the different indirect time periods (t1, t2) is incremented separately. Triple resonance experiments are the method of choice for the sequential assignment of larger proteins (> 150 AA). These experiments are called “triple resonance” because three different nuclei ( 1 H, 13 C, 15 N) are correlated. The experiments are performed on doubly labeled ( 13 C, 15 N) proteins.
Anatomy of a 2D experiment - continued After preparation the spins can precess freely for a given time t1. During this time the magnetization is labeled with the chemical shift of the first nucleus. During the mixing time magnetization is then transferred from the first nucleus to a second one. Mixing sequence utilize two mechanisms for magnetization transfer: scalar coupling (J-coupling) or dipolar interaction (NOE). Data are acquired at the end of the experiment (detection, often called direct evolution time); during this time the magnetization is labeled with the chemical shift of the second nucleus.
2D COSY (COrrelated SpectroscopY
) In the COSY experiment, magnetization is transferred by scalar coupling (J-coupling). Protons that are more than three chemical bonds apart give no cross signal because the 4 J coupling constants are close to 0. Therefore, only signals of protons which are two or three bonds apart are visible in a COSY spectrum (red signals). The cross signals between HN and H protons are of special importance because the
phi
torsion angle of the protein backbone can be derived from the 3 J coupling constant between them.
2D TOCSY (TOtal Correlated SpectroscopY)
In the TOCSY experiment, magnetization is dispersed over a complete spin system of an amino acid by successive scalar coupling (J-coupling). The TOCSY experiment correlates all protons of a spin system. Therefore, not only the red signals are visible (which also appear in a COSY spectrum) but also additional signals (green) which originate from the interaction of all protons of a spin system that are not directly connected via three chemical bonds. Thus a characteristic pattern of signals results for each amino acid from which the amino acid can be identified. However, some amino acids have identical spin systems and therefore identical signal patterns. They are: cysteine, aspartic acid, phenylalanine, histidine, asparagine, tryptophane and tyrosine ('AMX systems') on the one hand and glutamic acid, glutamine and methionine ('AM(PT)X systems') on the other hand.
TOCSY
H C H TOCSY
H C H TOCSY
H C H N H C H C O N H C C N H O H C C H O COSY NOESY COSY NOESY COSY
2D NOESY (Nuclear Overhauser Effect Spectroscopy)
The NOESY experiment is crucial for the determination of protein structure. It uses the dipolar interaction of spins (the nuclear Overhauser effect, NOE) for correlation of protons. The intensity of the NOE is in first approximation proportional to 1/r 6 , with r being the distance between the protons: The correlation between two protons depends on the distance between them, but normally a signal is only observed if their distance is smaller than 5 Å. The NOESY experiment correlates all protons which are close enough. It also correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure. This is the most important information for the determination of protein structures
What’s happening during the pulse sequence ?
RF pulse : I ( I ) I cos + I sin , and represent permutations of the x, y and z axes and is the pulse flip angle Example : I y (90I x ) I z I x (90I y ) -I z
Chemical shift : I x (w I tI z ) I x cos (w I t) + I y sin (w I t) I y (w I tI z ) I y cos (w I t) – I x sin (w I t) 2I z S x ( I x ) 2I z S x cos – 2I y S x sin 2I x S z (w I tI z ) 2I x S z cos (w I t) + 2I y S z sin (w I t) I z S z I y S z I x
J-coupling : I x ( J IS t2I z S z ) I x cos ( J IS t) + 2I y S z sin( J IS t) 2I y S ( J IS t2I z S z ) z 2I y S z cos ( J IS t) - I x sin ( J IS t) Example : I x ( J IS t2I z S z ) 2I y S z (t = 1/2J IS ) 2I y S ( J IS t2I z S z ) z - I x (t = 1/2J IS )
COSY : I: S: 90 90 x x t a b c a b : I x (wtI z ) I x cos (w I t) + I y sin (w I t) ( J IS t2I z S z ) [I x cos (w I t) + I y sin (w I t)] cos( J IS t) + [2I y S z cos(w I t) - 2I x S z sin(w I t)] sin( J IS t) b (90I x ) (90S x ) c : [I x cos (w I t) + I z sin (w I t)] cos( J IS t) + [-2I z S y cos(w I t) + 2I x S y sin(w I t)] sin( J IS t) I (COSY) x -2I z S y cos(w I t) sin( J IS t)
NOESY: 90 x I: t 1 90 -x m 90 x t 2 S: t a b c d e a b : I x (wtI z ) I x cos (w I t) + I y sin (w I t) ( J IS t2I z S z ) [I x cos (w I t) + I y sin (w I t)] cos( J IS t) + [2I y S z cos(w I t) - 2I x S z sin(w I t)] sin( J IS t) b c (90I x ) : [I x cos (w I t) + I z sin (w I t)] cos( J IS t) + [2I z S z cos(w I t) + 2I x S z sin(w I t)] sin( J IS t) c d :
?
HMQC : 180 x I: 90 x 90 x S: t a b c d e a b : I x ( J IS t2I z S z ) 2I y S z ( = 1/2J IS ) f b (90S x ) c : -2I y S y c (Dec CS) d : -2I y S y cos(w S t) + 2I y S x sin(w S t) d (90S x ) e : -2I y S z cos(w S t) + 2I y S x sin(w S t) e ( J IS t2I z S z ) f : I x cos(w S t) +2I y S x sin(w S t) HMQC: I x I x cos(w S t) ( = 1/2J IS )
t Anatomy of a 3D experiment 1 m t 2 MLEV t 3 FID Preparation Evolution Mixing Time Evolution Mixing Time Detection A three dimensional NMR experiment (see picture above) can easily be constructed from a two-dimensional one by inserting an additional indirect evolution time and a second mixing period between the first mixing period and the direct data acqusition. Each of the different indirect time periods (t1, t2) is incremented separately. Triple resonance experiments are the method of choice for the sequential assignment of larger proteins (> 150 AA). These experiments are called 'triple resonance' because three different nuclei (1H, 13C, 15N) are correlated. The experiments are performed on doubly labelled (13C, 15N) proteins.
What’s kind of the structural information that we can observe from NMR ?
Structural information
1. Interproton distances :
NOE
R
6
2. Dihedral angles :
J-coupling and Karplus equations
3. Chemical Shift Index (CSI) :
Chemical shift of H
, C
, C
, CO
4. Hydrogen bonding :
Amide proton exchange rates
• The distances between vicinal protons vary between approximately 2.15 and 2.90 A, and the exact values are determined by the intervening torsion angle. For instance, the distances d N (
i,i
) and d (
i,i
) are related with i and c i , respectively.
1
H-
1
H distance in Proteins
• Notation for
1
H-
1
H distances d d
N
(
i,j
)
d
NN
(
i,j
)
d
N
(
i,j
)
d
(
i,j
)
(i,j)
d(
H
i
, NH
j
) d(NH d(
H
i i
min{d( , , NH min{d(
H H H
i j
i
)
j
) , NH ,
H
j
j
)} )}
Karplus relations
• For structure determination of proteins the most important Karplus relations are
• • • •
3 J NH
3 J
3 J N
3 J C’
= 6.4 cos 2
q
= 9.5 cos 2
q
– 1.4cos
q
– 1.6cos
q
= -4.4 cos 2
q
= 8.0 cos 2
q
+1.2cos
q
– 2.0cos
q
+ 1.9
+ 1.8
+ 0.1
(a) Chemical shifts and secondary structure
NMR data with structural content : Chemical shifts (H
) down field
-sheet Random coil
-helix
(
sheet -
random coil ≈ 0.76 ppm)
Coupling constants (
3
J )
For L-amino acid : 3 J = 6.4 cos 2 (
– 60º) – 1.4cos(
– 60º) + 1.9
For D-amino acid : 3 J = 6.4 cos 2 ( 3 J > 9 Hz, 3 J < 4 Hz,
=
=
+ 60º) – 1.4cos(
1
20º
±
30º
3
0º
±
40º
+ 60º) + 1.9
13 C Chemical Shift Values Categorized According to Secondary Structural Assignment a-d Residue type e Ala (112) Cys (27) Asp (97) Glu (132) Phe (74) Gly (121) His (24) Ile (86) Lys (138) Leu (113) Met (36) Asn (71) Pro (53) Gln (61) Arg (65) Ser (88) Thr (105) Val (114) Trp (12) Tyr (43) Helix (DDS) Strand (DDS) Coil (DDS) 54.7
60.0
56.7
59.2
60.7
46.5
58.5
64.7
59.3
57.8
57.8
55.8
65.9
58.7
59.4
61.2
65.8
65.7
59.0
60.7
50.3
56.1
52.3
54.6
56.1
44.6
55.1
59.8
54.8
53.9
54.1
51.9
62.5
54.0
54.8
56.8
60.6
60.0
55.2
56.6
52.4
56.0
54.2
56.4
57.8
45.4
55.5
61.3
56.6
55.7
55.7
55.7
53.2
55.8
56.7
58.2
62.0
62.3
56.4
57.5
Spera (1991) (TSP) 52.3
56.9
54.0
56.4
58.0
45.1
61.3
56.5
55.1
55.3
52.8
63.1
56.1
56.1
58.2
62.1
62.3
57.7
58.1
Richarz (1978) (dioxane) 50.8
53.9
52.7
55.4
56.2
43.9
53.6
59.6
54.6
53.8
54.0
51.5
61.9
54.1
54.6
56.6
60.1
60.7
55.7
56.3
a Experimentally measured random coil values from Richarz and Wuthrich and from Spear and Bax are included for comparison. Data are given in ppm.
b The compounds (DDS, TMS, or dioxane) used in referencing the data are shown at the top of each column.
c To adjust DSS values to “old” dioxane standard, substract 1.5 ppm.
d To adjust DSS values to TSP, add 0.1 ppm.
e Total number of residues observed is given in parentheses. The data cover a grand total of 1572 amino acids.
Random Coil Chemical Shifts for Backbone Atoms in Peptides and Proteins a Residue Arg Ser Thr Val Trp Tyr Ala Cys Asp Glu Phe Gly His Ile Lys Leu Met Asn Pro Gln 1 H b 4.33
4.54
4.71
4.33
4.63
3.96
4.60
4.17
4.33
4.32
4.48
4.74
4.42
4.33
4.35
4.47
4.35
4.12
4.66
4.55
N 1 H 8.15
8.23
8.37
8.36
8.30
8.29
8.28
8.21
8.25
8.23
8.29
8.38
8.27
8.27
8.31
8.24
8.19
8.18
8.28
2 13 C 52.2
56.8
53.9
56.3
57.9
45.0
55.5
61.2
56.4
55.0
55.2
52.7
63.0
56.0
56.0
58.1
62.0
62.2
57.6
58.0
1 13 C 177.6
174.6
176.8
176.6
175.9
173.6
174.9
176.5
176.5
176.9
176.3
175.6
176.0
175.6
176.6
174.4
174.8
176.0
173.6
175.9
15 N 122.5
118.0
120.6
121.3
120.9
108.9
119.1
123.2
121.5
121.8
120.5
119.5
128.1
120.3
120.8
116.7
114.2
121.1
120.5
122.0
a Proton and carbon shifts are relative to DDS, nitrogen shifts are relative to NH 3 . Data are given in ppm.
b 1 H shifts were measured using the hexapeptide GGXAGG in 1M urea at 25C.
Wishart and Skyes, Methods Enzymol. (1994),
239
,363-392.
(b) COSY spectroscopy and its structural connectivities
(A) 1D 1 H spectrum (B) 2D COSY spectrum (C) Same as (b), contour plot
Eight areas containing different connectivities :
• a.
• b.
• c.
• d.
• e.
All nonlabile, nonaromatic amino acid side chain protons except H CH 3 of Thr, H H of Pro, and H H of Ser.
H CH 3 of Ala and H CH 3 of Thr.
H H of Val, Ile, Leu, Glu, Gln, Met, Pro, Arg, and Lys.
H H of Cys, Asp, Asn, Phe, Tyr, His, and Trp.
H H of Gly, H H of Thr, H H of Pro, H H and H H of Ser. • f.
• g.
• h.
Aromatic ring protons, including the four-bond connectivity 2H-4H of His and side chain amide protons of Asn and Gln Backone NH H.
CH 3 – e NH of Arg.
(A) 1D 1 H spectrum (B) 2D COSY spectrum (C) Same as (b), contour plot
(c) NOE spectroscopy and its Applications to Macromolecules
Nuclear Overhauser Effects (NOE)
• • • The NOE phenomenon is intimately related to spin relaxation. Analogous to the spin relaxation T frequency w o 1 and T , and the rotational correlation time c .
2 , the NOE varies as a function of the product of the Larmor Considering a pair of closely spaced spins
i
and
j
, connected by the vector or large spherical molecule. As a result of the collisions with the surrounding solvent and solute molecules, the thermal motions of these spheres consist of a random walk, which includes both translational and rotational movements. The relevant quantity for dipole-dipole relaxation and NOE is the rotational tumbling of the vector q ij
r
ij , and the concomitant time variation of the angle between
r
ij and
B
o .
r
ij , and located either in a small If the mobility of this vector is restricted to the overall rotations of the molecule,
r
ij will change orientation much more frequently in the small molecule than in the large molecule. For spherical particles of radius
a
in a solvent of viscosity estimated as , a correlation time characterizing the frequency range for these stochastic motions can be c = 4
a
3 /3
k
T
The NOE is a consequence of modulation of the dipole-dipole coupling between different nuclear spins by the Brownian motion of the molecules in solution, and the NOE intensity can be related to the distance r between pre-irradiated and observed spin by an equation of the general form NOE 1/r 6 *
f
( c )
f
( c ) is a function of the
correlation time
c , which accounts for the influence of the motional averaging process on the observed NOE. It seems to indicate that distance measurements with the use of NOE’s should be straightforward, provided that
f
( c ) can be independently assessed. In reality, a number of fundamental and technical obstacles tend to render quantitative distance measurements difficult. Thus, in all NOE experiments, and in particular in NOESY, processes other than NOE’s may also be manifested and can lead to falsification of apparent NOE intensities. Quite generally, because of the low sensitivity for observation of NOE’s, the accuracy of integration of line intensities is also limited by low S/N. Fundamental difficulties can then also arise when trying to correlate experimental NOE intensities with distances, for example, because of spin diffusion or the prevalence of intramolecular mobility in macromolecules.
In a small molecules, for example, a tripeptide or dinucleotide in aqueous solution, c is short relative to w o -1 (at 500 MHz, w o -1 = 3 x 10 -10 s). In this extreme motional situation, the frequency range covered by the rotational motion of r ij includes w o -1 and 2 w o -1 , which enables dissipative transitions between different spin states. In contrast, for macromolecules c is long relative to w o -1 , and the frequencies of the rotational motions are too low to allow efficient coupling with the nuclear spin transitions. Therefore, energy-conserving transitions of the type i i i j (cross relaxation) are favored.
• In the 1D experiments with relative line intensities I in the absence of NOE’s, the line intensities with NOE then become I = 1 + i / 2 j ( NOE factor ) • The 1 H{ 1 H} NOE, which is of prime interest for conformational studies, is +0.5 for the extreme motional narrowing situation.
For c , longer than approximately 1 x 10 -9 s it adopts a value of -1.0. For 13 C and 31 P {1H} the NOE factor is positive over the entire c range and becomes very small for long c . For 15 N {1H}, the NOE factor is negative throughout because of the negative value of very small and relatively of little practical importance in macromolecules.
. The NOE’s 1H{31P}, 1H{13C}, and 1H{15N}, are • In general, mechanisms other than dipole-dipole coupling with the preirradiated spin contribute to the T 1 relaxation. If T 1d (j) accounts for the dipolar relaxation between contribution to T 1
i
and
j
and T 10 for all other of spin i, NOE factor becomes ( i /2 j ) (T 1d (j) -1 /(T 1d (j) -1 + T 10 -1 ) • Accordingly, the NOE can be partially or completely
quenched
proximity of spin
i
in the presence of alternative, efficient relaxation pathways, for example, through to a paramagnetic center .
NOE and structural determination
• In principle, all hydrogen atoms of a protein form a single network of spins, coupled by the dipole dipole interaction. Magnetization can be transferred from one spin to another not only directly but also indirectly via other spins in the vicinity-an effect called spin diffusion. • The approximation of isolated spin pairs is only valid for short mixing time in the NOESY experiment. However, the mixing time cannot be made arbitrarily short because the intensity of a NOE is proportional to the mixing time. • In practice, a compromise has to be made between the suppression of spin diffusion and sufficient cross-peak intensities, usually with mixing time in the range of 40-80 ms. Spin diffusion effects can also be included in the structure calculation by complete relaxation matrix refinement, care has to be taken not to bias the structure determination by over-interpretation of the data.
NOESY: 90 x I: t 1 90 -x m 90 x t 2
•
Sequential distances
backbone protons or between a backbone proton and a are those between proton in residues that are nearest neighbors in the sequence. For simplicity, the indices i and j are omitted for the sequential distance; for example, d N (
i,i+1
) and d NN (
i,i+1
) d d N N N •
Medium-range
proton and a distances are all non sequential inter-residue distances between backbone protons or between a backbone proton within a segment of five consecutive residues.
•
Long-range backbone
that is i-j distances.
distances are between backbone protons in residues that at least six positions apart in the sequence, 5. All other inter-residue distances are referred to as long-range
Short ( < 4.5 A) Sequential and Medium Range 1 H 1 H Distances in Polypeptide Secondary Structures d Distance -helix 3 10 -helix N (i,i+1) 3.5
3.4
2.2
d N (i,i+2) 4.4
3.8
P 2.2
turn I a 3.4
3.2
3.6
turn II a 2.2
3.2
3.3
d N (i,i+3) 3.4
d N (i,i+4) 4.2
d NN 2.8
3.3
2.6
4.3
4.3
3.1-4.2
2.6
2.4
3.8-4.7
4.5
2.4
d NN (i,i+2) d N b 4.2
4.1
2.5-4.1
2.9-4.4
3.2-4.5
3.7-4.7
3.8
2.9-4.4
3.6-4.6
4.3
3.6-4.6
3.6-4.6
d (i,i+3) b 2.5-4.4
3.1-5.1
a For the turns, the first of two numbers applies to the distance between residues 2 and 3, the second to that between residues 3 and 4. The range between indicated for d N (i,i+3) corresponds to the distances adopted if 1 is varied between -180 and 180 .
b c 1 The ranges given correspond to the distances adopted by a -methine proton if is varied between -180 and 180 .
• The distances between vicinal protons vary between approximately 2.15 and 2.90 A, and the exact values are determined by the intervening torsion angle. For instance, the distances d N (
i,i
) and d (
i,i
) are related with i and c i , respectively.
Torsion angles for regular polypeptide conformations
Structure Hypothetical fully extended Anti-parallel sheet Parallel sheet Right-handed -helix Left-handed helix 3 10 helix Helix Torsion angle (degree) +180 (≡ -180) +180 -139 -119 -57 ~+60 -49 -57 +135 +113 -47 ~+60 -26 -70
(c) Distance d NN and torsion angles relationship – Ramachandran plot
Other NMR data for structure determination
• NOEs and scalar coupling constants are the NMR data that most directly provide structural information. Additional NMR parameters that are sometimes used in structure determination include hydrogen exchange data and chemical shifts, in particular hydrogen bond. 13 C . Slow hydrogen exchange indicates that an amide proton is involved in a • It was recognized that the deviations of 13 C (and, to some extent in 13 conformation: C 13 ) chemical shifts from their random coil values are correlated with the local backbone C chemical shifts larger than the random coil values tend to occur for amino acid residues -helical conformation, whereas deviations toward smaller values are observed for residues in b-sheet conformation. Such information can be included in a structure calculation by restricting the local conformation for a residue to the -helical or and structure is not perfect.
-sheet region of the Ramachandran plot, although care should be applied because the correlation between chemical shift deviation
Resonance assignment strategies for small proteins 1. Spin system identification :
DQF-COSY and TOCSY experiments 2.
Sequence-specific assignment :
NOESY experiment **For protein < 10 kDa, 2D homonuclear experiments may be sufficient for resolving overlapping NMR resonances.
DQF-COSY : Double-Quantum Filter-Correlation Spectroscopy TOCSY : Total Correlation Spectroscopy NOESY : Nuclear Overhauser Effect Spectroscopy
TOCSY
H C H TOCSY
H C H TOCSY
H C H N H C H C O N H C C N H O H C C H O COSY NOESY COSY NOESY COSY
D
etermination of
M
acromolecular
S
tructure by
M
ultidimensional
NMR S
pectroscopy
Spin-spin coupling constants in peptides
Spin-spin coupling constants, like chemical shifts, depend on chemical environment and are therefore of great use in structure determination.
J-coupling constants
C N H 35 140 C 55 H C
7 C 15 O N H 92 11 C H
C O
2D HSQC (Heteronuclear single quantun correlation) Spectroscopy
The natural abundance of 15N and 13C is very low and their gyromagnetic ratio is markedly lower than that of protons. Therefore, two strategies are used for increasing the low sensitivity of these nuclei: Isotopic enrichment of these nuclei in proteins and enhancement of the signal to noise ratio by the use of inverse NMR experiments in which the magnetization is tranferred from protons to the hetero nucleus. The most important inverse NMR experiment is the HSQC the pulse sequence of which is shown above. It correlates the nitrogen atom of an NHx group with the directly attached proton. Each signal in a HSQC spectrum represents a proton that is bound to a nitrogen atom.
(a) HNCO : H N (i) N(i) C'(i-1) C C N C C N C C H H O
(i-1)-residue
H H
i-residue
O
Inter-residues
(b) HN(CA)CO : H N (i) N(i) C
(i) C'(i) C C N H C H C O N H C C H O
Inter- (weak) and intra- (strong)
(c) HNCA : H N (i) N(i) C
(i-1) C
(i) C C N H C H C O N H C H C O
Inter- (weak) and intra- (strong)
(d) HN(CO)CA : H N (i) N(i) C'(i-1) C
(i-1) C C N H C C H O N H C C H O
Inter-residues
(e) CBCANH : H N (i) N(i) C
(i-1) C
(i-1) C
(i) C
(i) C C N C C N C C H H O H H O
Inter- (weak) and intra- (strong), C and C
(f) CBCA(CO)NH : H
(i-1) C
(i-1) C'(i-1) H
(i-1) C
(i-1) N(i) H N (i) H C H C N C C N C C H H O H H
Inter-residues, C and C
O
(g) HN(COCA)HA : H N (i) N(i) C'(i-1) C
(i-1) H
(i-1) C N H C C H O N H C C C H O (h) HN(CA)HA : H N (i) N(i) C
(i) H
(i) C C N H C C H O N H C C H O
(i) TOCSY-HSQC : H
(i) H
(i) H
(
i
)
H
N
(i) N
(
i
)
H C H H C H H C H H C H N C C N C C H H O H H O (j) HCCH-TOCSY : H
C H H
(i) C
(i) H
(i) C
(i) H
(i)
C
(i) H C H H C H H C H N H C C H O N H C C H O
Gly Ser Ala Lys Leu
Three-dimensional CBCA(CO)NH Spectrum 15 N 1 H 13 C
w
2 ( 13 C),
w
3 ( 1 H) projection of three-dimensional CBCA(CO)NH spectrum 13 C (ppm) 1 H (ppm)
Strip plot of 3D CBCANH spectrum of inhibitor-2(1-172)
119.75
126.13
122.38
119.56
121.82
115.06
I14 L15 K16 N17 K18 T19 118.06
115.81
115.91
S20 T21 T22 117.97
117.60
122.10
S23 S24 M25 15 N 20.00
30.00
* * * * *
40.00
13 C 50.00
* * * * * * * * * * * * *
60.00
* * *
70.00
*
7.93
8.25
8.24
8.32
8.29
8.15
1 H 8.32
8.24
8.12
8.29
8.29
8.24
ppm
I14 L15 K16 N17 K18 T19 S20 T21 T22 S23 S24 M25 15 N 172.5
* * * * * * * * * * * *
175.0
13 C 177.5
180.0
7.93
8.25
8.24
8.32
8.29
8.15
1 H 8.32
8.24
8.12
8.29
8.29
8.24
ppm
Methods for resolving overlapping NMR resonances 1. 2D/3D homonuclear NMR experiments : 2D-DQFCOSY, 2D-TOCSY, 2D-NOESY, 3D-NOESY-TOCSY 2. 2D/3D heteronuclear NMR experiments : 2D-
15
N-HSQC, 3D-
15
N-NOESY-HSQC and triple-resonance experiments (
1
H,
13
C,
15
N)
The flowchart of the protein structure determination from NMR data Protein in solution ~0.3 ml, 2 mM NMR spectroscopy 1D, 2D, 3D, … Sample preparation : protein isolation purification, characterization, cloning, isotopic labelling Sequence-specific resonance assignment Extraction of Structural information Calculation of initial structure using distance geometry Secondary structure Distances between protons (NOE), Dihedral angles (J coupling), Amide-proton Exchange rate, Chemical shifts index Structure refinement using molecular dynamics simulation Final 3D structures
2D NMR Spectroscopy
A two-dimensional NMR experiment involves a series of one-dimensional experiments. Each experiment consists of a sequence of radio frequency pulses with delay periods in between them. It is the timing, frequencies, and intensities of these pulses that distinguish different NMR experiments from one another. During some of the delays, the nuclear spins are allowed to freely precess (rotate) for a determined length of time known as the
evolution time
. The frequencies of the nuclei are detected after the final pulse. By incrementing the evolution time in successive experiments, a two dimensional data set is generated from a series of one-dimensional experiments
Correlation spectroscopy
is one of several types of two-dimensional NMR spectroscopy. Other types of two-dimensional NMR include
J-spectroscopy
,
exchange spectroscopy
(EXSY), and Nuclear Overhauser effect spectroscopy (NOESY). Two-dimensional NMR spectra provide more information about a molecule than one-dimensional NMR spectra and are especially useful in determining the structure of a molecule, particularly for molecules that are too complicated to work with using one-dimensional NMR. The first two-dimensional experiment,
COSY
, was proposed by Jean Jeener, a professor at Université Libre de Bruxelles, in 1971. This experiment was later implemented by Walter P. Aue, Enrico Bartholdi and Richard R. Ernst, who published their work in 1976
2D NOESY spectrum of 50 mM Gramicidin in DMSO-d6
2D NOESY spectrum of ethylbenzene
2D NOESY spectrum of aromatic region of 12,14 ditbutylbenzo[g]chrysene
Continuing the connectivity, we can assign H10 as 7.76 ppm H11 as 7.60 ppm and H13 as 7.86 ppm. In the opposite direction, H7 is at 7.59 ppm , H6 at 7.55 ppm, H5 at 8.62 ppm, H4 at 8.54 ppm, H3 at 7.44 ppm, H2 at 7.34 ppm and H1 at 8.17 ppm. Aromaric region shows connectivity and separation into four color-coded proton groups
2D TOCSY spectrum of ethylbenzene
1 H 1 H COSY Spectroscopy
1H-1H COSY (COrrelated SpectroscopY) is useful for determining which signals arise from neighboring protons, especially when the multiplets overlap or there is extensive second order coupling. A COSY spectrum yields through bond correlations
via
spin-spin coupling . If a homonuclear coupling is resolved in the 1D spectrum, a correlation will appear in the COSY but if no splitting is observed then no correlation is likely. Two and three bond and sometimes four bond correlations yield COSY signals.
2D COSY spectrum of ethylbenzene
2D COSY spectrum of aromatic region of 12,14-ditbutylbenzo[g]chrysene
There are separated into four color-coded proton groups showing connectivity. Using horizontal and vertical lines, it is possible to separate each group and follow its connectivity. The
blue
group of four protons is connected in the order 8.62 ppm to 7.55 to 7.59 to 8.56, the
green
group of four protons in the order 8.54 to 7.34 to 7.44 to 8.17 and the
red
group or two protons, that correspond to H9 and 10 because they are the only group of two protons expected to have a three-bond coupling constant (8.9 Hz), are at 7.76 and 8.32 ppm. The
yellow
group of two protons correspond to H11 and 13 because the coupling constant is small (1.9 Hz) and consistent with a four bond correlation.
HMQC NMR Spectrum of of codeine
This is a 2D experiment used to correlate, or connect, 1H and 13C peaks for directly bonded C-H pairs. The coordinates of each peak seen in the contour plot are the 1H and 13C chemical shifts. This is helpful in making assignments by comparing 1H and 13C spectra.
This experiment yields the same information as the older "HETCOR" experiment, but is more sensitive, so can be done in less time and/or with less material. This is possible because in the HMQC experiment, the signal is detected by observing protons, rather than carbons, which is inherently more sensitive, and the relaxation time is shorter. This so-called "inverse detection" experiment is technically more difficult and is possible only on newer model spectrometers.
Contour plot of the HMQC spectrum. Because it is a heteronuclear experiment, the 2 axes are different, and the plot is not symmetrical. Unlike a COSY spectrum, there are no diagonal peaks.
HMQC NMR Spectrum of of codeine Expanded aliphatic region:
3 5 7 8 9 10 133 128 120 113 91 66 11
13 C
59
1 H
3 and 5 3 and 5 7 and 8 7 and 8 10 9 11, 16, 18, 18' 12 56 13 46 14 43 16 40 17 36 18 20 none 13, 13', 17, 17' none 11, 16, 18, 18' 13', 17, 17' 11, 16, 18, 18'
CIEAKLTDTTTES (13-mer peptide) I 2 CH3 I 2 CH3 L6 CH3 T10 CH3 T9 CH3 T7 CH3 T11 CH3 1 / ppm 1. 00 I 2 H E3 H L6 CH L6 H A4 H K5 CH2 K5 H K5 CH2 E12 H 2. 00 E3 CH2 E12 CH2 D8 H K5 CH3 3. 00 S13 H 4. 00 A4- NH/ C H T10 H T9 H T7 H T11 H I 2- NH/ C H 8. 75 E3- NH/ C H E12- NH/ C H 8. 50 L6- NH/ C H T7- NH/ C T9- NH/ C D8- NH/ C H K5- NH/ C H S13- NH/ C T10- NH/ C H H H H T11- NH/ C H 8. 25 2 / ppm Fig 5-19 Peptide Den7 TOCSY spectrum at pH 5.0 and 50mM phosphate buffer 300μL 及 30μL D2O, and 298 K.
I 2 CIEAKLTDTTTES (13-mer peptide) 1 / ppm 4. 00 A4 E3 E12 L6 K5 T10 S13 T9 T7 T11 4. 25 4. 50 D8 4. 75 8. 75 8. 50 8. 25 8. 00 Fig 5-17 胜肽 Den7 DQF-COSY 光譜圖 , pH 5.0
的 50mM phosphate buffer 300μL 及 30μL D2O,298K 的條件下測得 2 / ppm
I 2 CIEAKLTDTTTES (13-mer peptide) 1 / ppm 4. 00 E3 L12 L6 A4 T7 T9 T11 4. 25 K5 T10 S13 4. 50 D8 8. 75 8. 50 8. 25 8. 00 2 / ppm Fig 5-18 胜肽 Den7 NOESY 光譜圖 , pH 5.0
的 50mM phosphate buffer 300μL 及 30μL D2O,298K,mixing time 為 450ms 的條件下測得
1 H 1 H TOCSY
1 H 1 H TOCSY (TOtal Correlated SpectroscopY also known as HOHAHA – HOmonuclear HArtmann HAhn) is useful for dividing the proton signals into groups or coupling networks, especially when the multiplets overlap or there is extensive second order coupling. A TOCSY spectrum yields through bond correlations
via spin-spin coupling.
Correlations are seen throughout the coupling network and intensity is not related in a simple fashion to the number of bonds connecting the protons. Therefore a five-bond correlation may or may not be stronger than a three bond correlation. TOCSY is usually used in large molecules with many separated coupling networks such as peptides, proteins, oligosaccharides and polysaccharides. If an indication of the number of bonds connecting the protons is required, for example in order to determine the order in which they are connected, a COSY spectrum is preferable.
The pulse sequence used in our laboratory is the gradient enhanced TOCSY. The spin-lock is a composite pulse and should be applied for between 20 and 200 ms with a pulse power sufficient to cover the spectral width. A short spin-lock makes the TOCSY more COSY-like in that more distant correlations will usually be weaker than short-range ones. A long spin-lock allows correlations over large coupling networks. Too long a spin-lock will heat the sample causing signal distortion and can damage the electronics of the spectrometer. The attenuation should be set so that the 90 ° pulse with will be less than 1/(4SWH) (SWH is the spectral width in Hz) and typically 1/(6SWH). An attenuation of 12 dB with a 50 W amplifier yielding a 90 ° μs is typical.
pulse width of 35
Spin-spin coupling constant (J) characterizes scalar interactions (through-bond) between nuclei linked via a small number of covalent bonds in a chemical structure.
If two nuclei couple with non-zero spin in the molecule having, say, spin I
1
and I
2
, then it is found that the resonance of spin I
1
is split into 2I
2
+1 lines of equal intensity and that of spin I
2
is similarly split into 2I
1
+1 lines. The line separations are equal.
The interaction is known as spin-spin coupling.
field independent And J is and is customarily quoted in hertz (Hz).
2D HSQC (Heteronuclear single quantun correlation) Spectroscopy
/ ppm 1
G149 G81 G16 G128 G68 G130 G42 G44 G47 G19 G67
110.00
S94 S151 S131 S95 S99 N18 S100 N12 S76 S98 N43 S75 N29 N11 N10 S94 D134 H32 T30 H108 S52 S74 S125 S97 S36 S34 S39 T66 S20 S135 T4 S103 S77 D111 H92 M83 Q91 Q8 V58 Q116 E150 V121 I144 I86 I78 E126 E60 E122 Q59 M118 L40 L37 A51 A7 A154 E23 A54 A57 L87 A90 L145 L65 A5 N102 Q50 Q46 Q130 E56 Q112 E62 H79 E61 E63 W152 N80 E84 D139 E141 E114 K148 D133 D120 M26 D89 E13 M146 R127 E48 Q33 E93 Q53 D143 Q31 R106 E14 H71 M136 E70 E132 M123 V64 L9 D82 E25 L55 K138 F119 L41 A6 W21 D155 L69 D155 I117 L88 L24 K85 L17 V22
115.00
120.00
125.00
9.00
8.50
8.00
7.50
7.00
/ ppm 2 The natural abundance of 15N and 13C is very low and their gyromagnetic ratio is markedly lower than that of protons. Therefore, two strategies are used for increasing the low sensitivity of these nuclei: Isotopic enrichment of these nuclei in proteins and enhancement of the signal to noise ratio by the use of inverse NMR experiments in which the magnetization is tranferred from protons to the hetero nucleus. The most important inverse NMR experiment is the HSQC the pulse sequence of which is shown above. It correlates the nitrogen atom of an NHx group with the directly attached proton. Each signal in a HSQC spectrum represents a proton that is bound to a nitrogen atom.
Six areas containing different connectivities
:
• a. NH; aromatics-NH; aromatics • b. NH; aromatics-
H;
H of Pro;
H of Ser and Thr • c. NH; aromatics-aliphatic side chains • d.
H
;
H of Pro;
H of Ser and Thr-
H
;
H of Pro;
H of Ser and Thr • e.
H;
H of Pro;