NMR - method suited to study biomolecules
Pavel Kadeřávek
1. NMR - introduction
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
1. NMR - introduction
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
NMR introduction
• magnetic moments of nuclei (spin) - 1H, 1^C, lbN, J1P
• Larmor frequencies uo = —jB (we need strong B)
• slow relaxation     polarisation transfer
Effect of electron density
-CH=0
-C=CH--CH2-0--CH2-C=0
-CH2-C-
(CH3)4Si H2
_I I
0 ppm 10 ppm 20 ppm 30 ppm
1950: Proctor, Yu
1H NMR spectrum of a protein
1956 - first NMR spectrum of a protein
Structure information
Nuclear Overhauser effect:        Spin-spin scalar coupling: distance torsion angles
j
1985: first protein structure solved by NMR
Residual dipolar couplings - Structure
information
1995: Prestegard J.H., 1996 Bax A.
1. NMR
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
Interaction: titration
figure copied from Charlier C. et al., J. Am. Chem. Soc, 2017, 139, 1219-12227
Interaction: titration
figures copied from publications:
Dicks et al., BMC Moleculart and Cell Biology, 2019, 20: 23 Waudby at al., Scientific reports, 2016, 24826
Interaction of small molecule with a large
molecule
selective saturation
Wf'm  |A h H h
II II II I it li 11 1 , lAa
off-resonance on-resonance difference spectra
copied from publication: Addino Viegas et al., J. Chem. Educ, 2011, 88, 990-994
1. NMR
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
Conformational Exchange = Structure
Rearrangement
®c g e g
gggg@ g © qö
pa- pb = 1 - pa
/ys—ms motions
kex < Qa - Q
a -
Rex(kex, ^a> ^b> Pb)
A
B
k
ex
G © ßö
pa- pb = 1 - pa
/ys—ms motions
kex > Qa - Q
a -
Rex(kex, ^a> ^b> Pb)
A
B
k
ex
G © ßG
^a ^obs
b
pa> pb = 1 - pa
Conformational Exchange = Structure
Rearrangement
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Chemical Exchange Saturation Transfer
Residual Dipolar Coupling
Vallurupalli P. et al., Proc. Natl. Acad. Sei., 2007, 104, 18473-18477
1. NMR
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
RNA polymerase
DNA
• Gram positive bacteria Transcription                9 Bacillus subtilis
RNA polymerase
• RNA polymerase core:
_.. _ ol ol 3 3 (jj
RNA ^ ^
Translation Ribosom
t
PROTEIN
S subunit
important for virulency
2 domain protein:
N-terminal domain: structured C-terminal domain: disordered
MGIKQYSQEE LKEMALVEIA HELFEEHKKP VPFQELLNEI ASLLGVKKEE LGDRIAQFYT DLNIDGRFLA LSDQTWGLRS WYPYDQLDEE TQPTVKAKKK KAKKAVEEDL DLDEFEEIDE DDLDLDEVEE ELDLEADDFD EEDLDEDDDD LEIEEDIIDE DDEDYDDEEE EIK
residue number
1. NMR
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
Intrinsically disordered protein (IDP)
• intrinsically disordered protein (IDP)
- lack of stable 3D structure
- high flexibility
- structural adaptability
- polyfunctionality
figure copied from:
softsimu,blogspotir/2013/preformed-structural-elements-in-long.html
Intrinsically disordered protein (IDP) - challenges
Free energy landscape
B c
Rigid protein
Multiple conformations
IDP
figure copied from:
M. Bruscale, B. Schuler, B. Samori, Chem. Rev., 2014, vol. 114, 3281
Intrinsically disordered protein (IDP) - challenges
• complex motion - distribution of timescales
• NMR relaxation at different magnetic fields - sensitive to various frequencies
• low frequencies =>■ low Bo = low resolution
10
r/ns
High-resolution relaxometry
Br
Bore of the spectrometer
Area of homogeneou high magnetic field
Sample
High-field probe
High-resolution relaxometry
Bore of the spectrometer
Area of homogeneou high magnetic field
Sample
High-field probe
High-resolution relaxometry
Bore of the spectrometer
Area of homogeneou high magnetic field
Sample
High-field probe
High-resolution relaxometry
Bore of the spectrometer
Area of homogeneou high magnetic field
Sample
High-field probe
High-resolution relaxometry - device
NMR spectrometer Sample shuttle tube   Triple resonance probe
pneumatic shuttling: 0.5 m in ~120ms
Charlier C. et al., J. Am. Chem. Soc, 2013, 135 (49), 18665-18672
delta subunit: longitudinal relaxation rate
10
14.10T
C/3
DC
8 h
6 h
4 h
2 h
•** *
* *** *****
0
80        90        100       110       120 130
residue number
140
150
160
170
delta subunit: relaxometry relaxation rate
		14.10T i—i—i
-		4.00 T i—i—i
		
**** *** **** i	*** * i             i             i i	*** ***** i           i i
80        90        100       110       120       130       140       150       160 170
residue number
delta subunit: relaxometry relaxation rate
10
14.10T i—i—i 4.00 T i—i—i 1.00 T i—i—i
8 h
6 h
I    T "
111 %*%
I*
4 h
2 h
*** ***** %
0
80        90        100 110
120 130 140 150 residue number
160 170
delta subunit: distribution of motions
0.6 0.5
CM 0.4
<
t- 0.3 0.2 0.1 0.0
< 10"1 ns
1
80    90   100  110  120  130  140  150  160 170 residue number
CO
<
0.6 0.5 0.4 0.3 0.2 0.1 0.0
- 10"1 ns
ill il,i.lii,iIIII1M
80    90   100  110  120  130  140  150  160 170 residue number
0.6 0.5 0.4 < 0.3 0.2 0.1 0.0
-10° ns
11
ml
80    90   100  110  120  130  140  150  160 170 residue number
0.6 0.5 0.4 < 0.3 0.2 0.1 0.0
> 10° ns
1
As
80    90   100  110  120  130  140  150  160 170 residue number
81____DQLDEE TQPTVKAKKK KAKKAVEEDL DLDEFEEIDE DDLDLDEVEE
ELDLEADDFD EEDLDEDDDD LEIEEDIIDE DDEDYDDEEE EIK
1. NMR
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
Metabolomics
• investigation of metabolites in biological fluids
• substrates and products of enzymatic reactions
• cofactors or regulators
copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404
Metabolomics
• dependence of relaxation rate on rotational diffusion
• Stokes law =^> size of the molecule     free x bound
10
5
0.01
0.1 1
Magnetic field (T)
10
12 10 ■P 8 t£ 6
4 -
Alanine TSP
■ _J—L
0.1 1 Magnetic field (T)
copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404
Metabolomics
106 10s 10"4
Concentration (mol/L)
copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404
Metabolomics
competition between ligands
0.1 1 10 0.1 1 10
Magnetic field (T) Magnetic field (T)
copied from publication: Ziqing Wang et al., J. Am. Chem. Soc, 2021, 143, 9393-9404
1. NMR - introduction
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics
of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
TOCSY
TOCSY = total correlation spectroscopy:
• information about bonds
• requires high irradiation suppressing effects of chemical shift
• stronger magnetic field = stronger irradiation
C62 C61
CY Ca
Ca
C3
Cy C61C62
TOCSY
50		•
E Q_ Q_ U 100 m i—i		
i # H K)		
150	High-field TOCSY • *	
150 100 50
(5,-13C (ppm)
Two-field NMR spectrometer
NMR spectrometer Sample shuttle tube   Triple resonance probe
pneumatic shuttling: 0.5 m in ~120ms
Cousin S.F. et al., Phys Chem Chem Phys, 2016, 18 (48), 33187-33194
TOCSY at two-fields
				š           j tí
		• ♦>		• *
50	» *	o 4»		6          o set 0 •
(ppm)				
u 100 m i—i				
1	•			t i 1 *
150	2F • •	-TOCSY		O Q •                 a                   o t>*
T-1-1-1-1-1-1-I-1-1-1-1-1 I-1 I
150 100 50
Ô7-13C (ppm)
TOCSY at two-fields
180
150 140 r52-13C (ppm)
/ys—ms motions
kex > Qa - Q
a -
Rex(kex, ^a> ^b> Pb)
A
B
k
ex
g q q 0©
g © qQ
^a ^obs
b
pa> pb = 1 - pa
Exchange effects suppression at two-fields
1. NMR - introduction
2. Studies of interactions
3. Conformational exchange
4. Investigation ps-ns dynamics of structured proteins
5. High-resolution relaxometry - investigation of ps-ns dynamics of IDPs
6. High-resolution relaxometry - metabolomics
7. Two-field NMR
8. Dissolution dynamics nuclear polarisation (dDNP)
Dissolution dynamics nuclear polarisation
(dDNP)
Temperature (K)
copied from publication: Bertrand Plainchont et al., Anal. Chem., 2018, 90, 3639-3650
Dissolution dynamics nuclear polarisation
(dDNP)
r
Magnetic Tunnel
copied from publication: Bertrand Plainchont et al., Anal. Chem., 2018, 90, 3639-3650
Dissolution dynamics nuclear polarisation
(dDNP)
copied from publication: Sami Jannin et al., J. Mag. Reson., 2019, 305, 41-50