NUCLEAR MAGNETIC RESONANCE:
ADVANCES IN CHEMISTRY AND
BIOLOGY
Ryszard Stolarski
Division of Biophysics
Institute of Experimental Physics
Faculty of Physics
Warsaw University
NMR in biophysics, chemistry, biology and medicine
• Stern-Gerlach experiment (1921)
• Resonance method and determinations of nuclear moments by
atomic- and molecular - beam experiments (1938)
• Detection of nuclear magnetic resonance in bulk matter (1946)
Nobel prize in physics for F. Bloch (Stanford) and E. M. Purcell
(MIT) in 1952 r.
• Fourier transformation (1960/1970) and two-dimensional NMR
of small proteins and nucleic acid fragments (1970/80)
Nobel prize in chemistry for R. Ernst (ETH, Zurich) in 1991
Nobel prize in chemistry for K. Wüthrich (ETH, Zurich) in 2002
• Nuclear magnetic resonance imaging (MRI) of intact bodies
Nobel prize in physiology and medicine for P.C Lauterbur
(Stony Brook) and P. Mansfield (Nottingham Univ.) in 2003
• Multidimensional (3D and 4D NMR) in structural determinations
of proteins and nucleic acids up to ~40 kDa (1990/2000)
• TROSY and CRINEPT experiments; spectrometers with magnets
over 18 T (800 MHz); cryoprobes (2000 - )
High resolution NMR
Liouville-von Neumann equation
dρ
i
 [H, ρ]  R(ρ  ρ0 )
dt
 - density matrix
R - relaxation superoperator (relaxation
times T1 i T2)
N
N
interaction with the
exciting field B1
H    jIzj   hJ(i, j)Ii  I j  B1( t )   I j
j1
i j
j1
chemical shifts j
scalar couplings
N
N
j1
j1
ρ( t p )  exp( iB1t p  Isj )  ρ  exp(iB1t p  Isj );
N

Mx ( t )  n      Tr   Ikx ρ( t )
k 1

N

My ( t )  n      Tr   Iky ρ( t )
k 1

s = x, y
Transformation of the
density matrix after B1
pulse of tp duration
Observables: x- and y-components of
magnetization of N nuclei  in a molecule
of concentration n in solution
Applications of NMR
"Broad line" NMR:
condensed matter
investigations
(biological
membranes)
Studies of small molecules in
solution: structure, dynamics,
interactions, and physicochemical properties
Medical diagnostics:
magnetic resonance
imaging” (MRI)
Studies of biochemical
processes in intact
cells:
- in vivo NMR,
- in cell NMR
Quantum computers
Determination of structures
and dynamics of biopolymers
in solution
Biopolymers - nucleic acids and proteins
Genome
full set of genetic
information in the body
Proteome
full set of proteins
coded by the genes
Genomics
sequencing of DNA and
identification of the genes
Proteomics
complete characteristics
of the proteome
Transcription
(mRNA synthesis)
Translation
(protein synthesis)
mRNA
DNA
nucleus
Protein
cytoplasm
Gene expression
Aims of structural proteomics
• High throughput determination of structures of most proteins
coded by sequenced genomes
• Molecular mechanisms of interaction of proteins with ligands.
• Sequence - structure - activity relationship for groups of proteins
interacting in a metabolic pathway
• Drug design; choice of suitable „targets” for chemotherapy
Structural parameters of proteins
NMR RESONANCE ASSIGNMENT: chemical shifts j, scalar couplings
J(i,j), and nuclear Overhauser effect (NOE)
rij
STRUCTURE
LOCAL
• Scalar coupling constants J(i,j)
Dihedral angles 
• Proton NOEs
Interproton distances rij
• Chemical shifts j
Secondary structure
(TALOS)
GLOBAL
• Residual dipolar couplings
Mutual orientations of
the molecular fragments
Protein structure in solution by multidimensional NMR
5 structures of BPTI (58 amino
acids) by 2D NMR with the
X-ray structure
20 structures of yeast eIF4E
(217 amio acids) by 3D NMR
 helix
 sheet
Wagner G. et al., J. Mol. Biol. (1987) 196, 611-639
Matsuo H. et al., Nature Struct. Biol. (1997) 4, 717-724
1D NMR
Single pulse experiment
FID signal
S   Mxy t  exp it dt
equilibrium
detection
pulse duration (s): t p 
pulse phase: 

2B1
1D spectrum of BPTI
Wüthrich K. et al., J. Mol. Biol. (1982) 155, 311-319
2D NMR
1H,1H-NOESY
pulse sequence
Signal:
S12  
  Mxy t1t 2  exp i1t1  exp i2 t 2 dt1dt 2
1H
equilibrium
evolution t1
mixing
detection t2
1 (1H)
1 (1H)
2D spectrum of BPTI
2 (1H)
Wagner G & Wüthrich K, J. Mol. Biol. (1982) 155, 347-366
2 (1H)
3D NMR
{15N/13C},1H,1H-HMQC-COSY
3D protein spectrum
and its 2D cross-section
1H
15N
or 13C
equilibrium
evolution t1
constant 
evolution t2 detection t3
constant 
Signal:
S123  
  Mxy t1t 2 t 3  exp i1t1  exp i2 t 2  exp i3 t 3 dt1dt 2dt 3
Oschkinat H. et al., Angew. Chem. Int. Ed. Engl. (1994) 33, 277-293
Functional Magnetic Resonance Imaging (fMRI)
MEDICINE
"PHILOSOPHY"
An fMRI Investigation of
Emotional Engagement
in Moral Judgement
Joshua D. Greene, R. Brian Sommerville, Leigh E.
Nystrom, John M. Darley, Jonathan D. Cohen
Science (2001) 293, 2105-2108
Magnetic resonance image of a transverse
slice of a monkey head; contrast based on
blood microcirculation
Geoffrey Sobering, Science (1990) 250
Electric charge distribution
in the 7-methylguanine ring of cap
O
*H
N
N
+
H2N
?
CH3
N
N
O
H
H
H
CH2
O
P
O
P
O
P
O
B
CH2
O
OH HO
H
O
O
O
O-
O-
O-
H
H
H
H
H
O
-O
cap structure
OCH 3
P
O
O
mRNA
Examples of fitting the NMR signals
using trial values of the couplings
JC2-N2 = -23.4
JC2-N9 = -3.7
19375
19400
19425
19450
20000
20050
20100
JC2-C4 = 7.8
19475
JC6-C5 = 87.9
JC6-N7 = -7.9
19950
C2 GTP
JC2-N1 = -14.6
JC2-N3 = -3.0
C6 GTP
JC6-C4 = 12.9
JC6-N1 = -5.9
JC6-C8 = 6.8
JC6-N9 = -1.3
20150
C8 m7GTP
JC8-N9 = -18.4 JC8-N7 = -18.4
17525
17550
17575
17600
17625
Changes of the NMR parameters
due to methylation at N7 of guanine:
shielding constants () and reduced couplings (1K)
1
K(m7GTP) - 1K(GTP)
1
K(m7Gua) - 1K(Gua)
B
C4-N9
C6-N1
C8-N9
H8-C8
C8-N7
C5-N7
C5-C6
C4-C5
C4-N3
H2-N2
C2-N2
C2-N3
H1-N1
C2-N1
*
-20
0
20
40
K [ x 10 -19 T2/J ]
60
80
Changes of the calculated atomic charges
due to methylation at N7 of guanine
Hirshfeld charges
ESP charges
Mulliken charges
N9
C8
N7
C6
C5
C4
N3
N2
C2
N1
-0.4
-0.2
0.0
0.2
q [au]
0.4
0.6
Conclusion:
localization of the net positive charge at N7
O
H1
H
N2
H
N1
C2
C6
N3
O
C5
C4
N7
C8
N9
R
guanosine
H1
H8
H
N2
H
N1
C2
C6
N3
+
C5
C4
CH3
N7
C8
N9
R
7-methylguanosine
H8
"Charge distribution in 7-methylguanine regarding cation-
interaction with protein factor eIF4E"
Biophysical Journal 85, 1450-1456, 2003
Division of Biophysics
Katarzyna Ruszczyńska-Bartnik, PhD student
Janusz Stępiński
Edward Darżynkiewicz
Institute of Organic Chemistry, PAS
Krystyna Kamieńska-Trela
Institute of Biochemistry and Biophysics, PAS
Jacek Wójcik