Nuclear Magnetic Resonance
Lukáš Žídek
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Principles of NMR
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I
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lecular rotation
reproduced from M. H. Levitt: Spin Dynamics
nuclear spin
flow of electrons induced field
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x
reproduced from M. H. Levitt: Spin Dynamics
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agnetic moments
lecular rotation
reproduced from M. H. Levitt: Spin Dynamics
nuclear spin
flow of electrons induced field
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x
reproduced from M. H. Levitt: Spin Dynamics
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Fourier transformation of ideal signal.
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rons
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• Solvent (water) suppression
• Simplification of spectra
• Resolution improvement
• Obtaining chemical/biological information structure, dynamics, interactions
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Transmitter on
Transmitter off Receiver on
Receiver off
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mm
R experiments
HSQC
H
N
I I ll I    I I I I II I
x-xj.   f * y
II I I I
GARP
gra
i li i
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< □ ►   4 ^ >  4 = t   4 = >      =     ^0 Q, O
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R experiments
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R experiments
10 5 0
ô(1H)/ppm
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9.0 8.5 8.0 7.5 7.0 6.5
i   i   i   i   i   i   i   ■   i   i   i   i   i   i   i   i   i   ■   i   i   i   i   i   i   i   i   i   ■   i i
0.5H
1.0H
E   1-5H
Q. Q.
2.0H
2M
3.0H
C02-15N : 113.721 (ppm)
0
o
O
■0.5
1.0
■1.5
■2.0
■2.5
■3.0
9.0
8.5
8.0
-i-1-1-
7.5
7.0
6.5
C03-1H (PPm)
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Biomolecular applications
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• No special sample requirements
• Low energy non-destructive x low sensitivity
high concentration, high magnetic field, isotope labeling long measurement time
• Atomic resolution
o Many atoms described by single measurement high information content x complexity of data correlated spectroscopy, selective labeling Assignment of spectra is demanding
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JCH-R [ C=0
-A"
H^—SN
^CH-R' C=0
/CTI
0
e 0
3
g
3
1       13      133      1332      13322      113322      4113322 44113322
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in NMR spectrum
3
2R2		t:	1/Ä2 1
	\		
ft —y structure
fľ2 —> dynamics
Area —> (relative) concentration
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ser effect
reproduced from M. H. Levitt: Spin Dynamics
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ser effect
_S_ = /A>ef\6 Sref     V r )
Calibration:
Reference protons distance
geminal in methylene H-C-H       0.17 nm
vicinal in an aromatic ring H-C=C-H 0.25 nm meta in an aromatic ring    H-C=CH-C-H   0.42 nm
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<D 20 '§> 15
8 ig
LU 5
O -5
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xperiments
3
			I/R2
	í		
216
D.M. Korzhnev et al. / Progress in Nuclear Magnetic Resonance Spectroscopy 38 (2001) 197-266
a
1H       III 1	§ 1 §	1 1
cj>l y 15N   I       1       1Tb 1Xb 1		if!    <()3 (|)4 |ťb| t1/2   Tb    t1/2 J
g1     g2     g2    g3     g3 g4 Gz ------		g5               g6 g7 n     1 n
RiP 15Nf		T
		
j Ta  j Ta   j Ta   j  Tc I
y
I I I
Acquisition
Broadband decoupling
g7     g7     g8 g8
99
JL
2N
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I
ration
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• Does it bind?
How many molecules? Stoichiometry
In how many steps? Mechanism
• Where? Structure
• How strongly? Affinity
• How fast? Kinetics
CD
CD
CD CD
1 1	
	AG*off \ /
Jag ,	Ay
	
Free Bound Binding coordinate
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• Ligand
saturation transfer difference (STD), transferred NOE features of bound-ligand reflected in free-ligand spectra not limited by the size of the protein
• Protein
usually more structural details
H
H
selective saturation
i
±1
off-resonance
on-resonance
difference spectra
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k,,,, = 0.2 s'
Kd = koff/kon=0.01 Cp
Ato(ligand) = 200 rad s"1 Ato(protein) = 200 rad s"1 R2(free ligand) = 2 s"1 R2(bound ligand) = 10 s"1 R2(free protein) = 10 s"1 R2(bound protein) = 10 s"1
0.0 0.5 1.0 1.5 2.0 2.5 3.0
CL/Cp
free ligand
Titration with ligand aliquots of cL = 0.5 cP:
1
1
1
1
JL
JL.
JU
JU
1
X-k.
1
free protein
<□►   4 ^ >  4 = t   4 = >      =     ^0 Q, O
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k = 0.5 x 103s1
k= 1.0x 10V
i—
-3 kHz
0
k = 2.5x 103s~1
k = 3.5x 103s1
k = 6.3x103s~1
—i
+3 kHz
Q/2%
reproduced from M. H. Levitt: Spin Dynamics
k = 6.3 x 103s1
k= 10 x 103s1
k = 20x 103s1
k = 30 x 103s1
A
k = 50x 103s1
i-1-1
-3 kHz       0       +3 kHz
£1/2%
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ion experiments
1H
2H
_LL1 I I
SPINLOCK
II I
y 41
11 n
1
o2
A.    ■ A.
WALTZ-16
1
II I
WALTZ-1S
1
91    g2   g2 g3
g4   g5    g5 g6
g7 g8 g8
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Interacting molecules:
• Receiver domain of plant sensory histidine kinase CKI1 (from Arabidopsis thaliana)
Mg
2+
ions
fosmotic stress
_' cytokinin
AHK1
cytokinin cytc
A-fAAAAAAAAAAl.44jlÍAAAAAAAAAAAWA4AiAAAAA!-A-U4AiAAlAAj aIaAAAAAAAAAAAAAAAAAAAAAAAAA'IAAAAA
\   |AHK3 /
AHK4
f
aaaaaaaaaaaaaaaaaaaaaaaaiaiaJaIaaj
Jit II
cytoplasm
AHP1-6
O—O
o—o o—o o—o o—o
0—0
o—o o—o
endoplasmic reticulum
. ethylene — ethylene
ooo^oo
•o ^ mo-
LI nucleus v
AHHo-y, 15-1/   ^^Vcj AAAAAAAAAAAAAlMAJAyAAAAAAl^AlMAAAAAAAAAA
•  -        % Tilt
target proteins Oj jjO
n
target proteins
EIRS1
ARR1,2,10-14,18-21
Pekařova et al., Plant J. 67 (2011) 827 Otrusinova et al., J. Biol. Chem. 292 (2018) 17525
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0.4
0.3 :
Q. Q.
OvJ
OvJ
I
to
LO OJ
+
OJ
Z
to
0.2 F
0.1 ŕ-
0.0 :
Free + Mg2+ ^ Bound
-j-1-1-1-1-r-	-1-1-1-1-j-1-1-1-1-r 3     8 ß	-1-1-1-1-j-1-1-1-1-r	—i-1-1-1-j—
			-
			■
0	10	20	30
cMg2+ / m M
Kd = 0.43 ± 0.06mM
(data for Q92)
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Interacting molecules:
• Mouse major urinary protein I
o male pheromone 2-sec-butyl-4,5-dihydrothiazole (estrus synchrony and puberty acceleration in females)
Zidek et al., Biochemistry 38 (1999) 9850
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i
1
I
a
t-H
i
s
					• ř
1.00	— ■ \	T t	-A-		f -A—
0.80	\	t t » f i r v I		#	
0.60		m » 'A			
0,40		M			
0.20		f                           1 t t                              * > /                                 v i			
0.00	i t r -f-		i	i	
0.00   0.50    1.00    1.50   2.00 2.50
Molar ratio of added pheromone to total protein
stoichiometry = 1.0 ± 0.1 /xM
Too strong for NMR
Determined by equilibrium diffusion/gas chromatography
ft
1
O
I
ft
o
0.00
0.00       1.00      2.00 3.00 [free pheromone] (pM)
4.00
Kd = 1.3±0.1/iM
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MUPI.pdb
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OE
A
B
D
13C.1H<__>1H.12C
.í	♦		pi	8a$ 9a/b			
	r						
			*			5«	
n	t	4$	#				4*'
° B
o £X
-n
o .y
x:
U
U
CM
O ffi
cm
Figure 7: Representative strips from 13C Fl-filtered, F3-edited NOESY—HSQC spectra of free (right strips) and 2-^c-butyl-4,5-dihydrothiazole-complexed (left strips) rMUP-L The strips were taken from the 3D spectra at chemical shift values corresponding to (A) Leu 40<5i, (B) Leu 105du (C) Tyr 120e2, and (D) Tyr 84(32. The NOE cross-peaks are labeled with the corresponding ligand proton numbers.
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OE
Table 3: Intermolecular NOEs between 2-sec-Butyl-4,5-dihydrothiazole and MUP-P
ligand
dihydrothiazole sec-butyl chain protons^ ring protons^
protein	9a/b	8a	8b	7a	7b	7a77b'	5	5'	4 4'
Leu 42Ó1	wc	x	x	m	x	md			
Leu 42Ó2	m	s				m			
Ala 103/3	vsd		wc	wc	x	x			
Leu 54ói	X	wc		x	m'	m			
Leu 54ó2	X	wc	sd	x	x	x			
Tyr 120e2	m	m							
Phe 90Ó2	m			wc					
Phe 90e2						m			
Phe 56e2	w				md	m			
Phe 56£	m			wc		m	md		md
Leu 105ói	x			x	x	x	w	m	m w
Leu 40ói	x			x			m	m	s s
Val 82y!	x	x	x	x	x	x	m	m	
Met 69e	x	x	x	x	x	x	s	w	w m
Tyr 84Ó2							m	wc	
a Strength of the NOEs is expressed in a semiquantitative manner (vs, very strong; s, strong; m, medium; w, weak; and x, obscured by background). b The symbols a and b in the proton labels refer to individual sec-butyl spin systems and diastereotopic protons are distinguished with a prime as indicated in Figure 6B. c Possible weak signal obscured by a close intense NOE peak. d Medium or intense peaks close to an area of high background.
•<[fj1^ < ^ >     ^    ^0 Q, O
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<□►   4 ^ >  4 = t   4 = >      =     ^0 Q, O
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