Nuclear Magnetic Resonance in Structural Biology

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Nuclear Magnetic Resonance in Structural
Biology Part I the physical principle the
spectrometer the NMR spectrum the
applications (what you can do) the
sample the limits (what you cannot
do) protein structure calculation
references
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Nuclear Magnetic Resonance in Structural
Biology Part II Journal Club A zinc clasp
structure tethers lck to T cell coreceptors CD4
and CD8. P.W Kim, Z.J. Sun, S. C. Blacklow, G.
Wagner, M. J. Eck Science 301(19 Sept)
1725-1728 (2003) (the PDF file can be downloaded
from www.sciencemag.org)
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The physical principle
nuclear spin, I½
Energy
-½
n gB/2p
B (magnetic field)
½
At B11.7 T (tesla, 104 Gauss) the resonant
frequency for 1H is 500 MHz (earth magnetic field
30-60 mT magnetic stirrer 0.1 T) In NMR,
excitation is achieved through short (ms)
electromagnetic (radio-frequency) pulses. The DE
is very small (10-2 cal) ? low population
difference ? low sensitivity
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The spectrometer
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The NMR spectrum In biomolecules (proteins,
nucleic acids, peptides, oligosaccharides, ...)
the information available is contained in the
NMR-active nuclei and limited by their natural
abundance and sensitivity 1H (99.98) 12C
(98.93) not active 14N (99.63) not
detectable 16O (99.76) not active 32S (94.93)
not active 31P (100) limited use resonant
frequency (chemical shift, ppm) signal
intensity J-couplings (spin-spin interactions
through covalent bonds) lineshape
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Other useful nuclei used in NMR
Frequency (MHz) at 2.3488
sensitivity (relative)
sensitivity (absolute)
isotope
I
abundance()
1H 1/2 99.98 1.00 1.00
100.000 2H 1 1.5x10-2 9.65x10-3
1.45x10-6 15.351 13C 1/2 1.108
1.59x10-2 1.76x10-4 25.144 14N 1
99.63 1.01x10-3 1.01x10-3 7.224 15N
1/2 0.37 1.04x10-3 3.85x10-6
10.133 19F 1/2 100 0.83
0.83 94.077 23Na 3/2 100
9.25x10-2 9.25x10-2 26.451 31P 1/2
100 6.63x10-2 6.63x10-2
40.481 113Cd 1/2 12.26 1.09x10-2
1.33x10-3 22.182
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The chemical shift
The resonant frequency of a certain atom is
called chemical shift. For convenience, the
chemical shift is expressed as follows
Advantages more compact annotations indepen
dent on the spectrometer field In practice, the
1H chemical shifts are in the range 0-10 ppm
The chemical shift depends on the atom type
(NH, aliphatic CH, aromatic CH, ...) the amino
acid type (Ala, Phe, ...) the chemical
(spatial) environment
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The 1D spectrum of a peptide
CH2
O
H
N
C
N
C
H
H
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The assignment problem Which resonance
corresponds to which atom?
CH3
H3C
through-bonds magnetization transfer
(J-couplings) through-space magnetization
transfer (NOE)
H
O
H
C
N
C
N
C
H
H
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The 2D spectrum The information contained in 1D
spectra can be expanded in a second (frequency)
dimension ? 2D NMR In a 1D experiment a resonance
(line) is identified by a single
frequency NH(f1nh) In 2D spectra, a resonance
(cross-peak) is identified by two different
frequencies NH (f1nh, f2ha) NH (f1nh,
f2ha) Usually, the second frequency depends on
how the NMR experiment is designed.
f2
f1
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The 2D spectrum of a peptide (DQF-COSY) Ac-GRGGFGG
RG-NH2
Ph
CH2
O
H
N
C
N
C
H
H
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The 2D spectrum of a peptide (TOCSY) Ac-GRGGFGGRG-
NH2

CH2
CH2
CH2
O
H
N
C
N
C
H
H
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The 2D spectrum of a peptide (ROESY)
Ac-GRGGFGGRG-NH2


CH2
O
N
C
N
C
H
H
H
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The NMR experiment as a black box ?
sample
NMR spectrum
?
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The 2D spectrum of a protein (NOESY)
Practical applications of 2D homonuclear NMR are
limited by peak overlap.
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From 1D to 2D and 3D NMR
1D
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The NMR spectrum In biomolecules (proteins,
nucleic acids, peptides, oligosaccharides, ...)
the information available is contained in the
NMR-active nuclei and limited by their natural
abundance. We can add information to the system
replacing inactive/undetectable nuclei with
active ones. This is called labeling. In fact,
it should be called isotopic enrichment or
isotope abundance reversal. These are naturally
occurring isotopes! 1H (99.98) 12C (98.93) not
active ? 13C 14N (99.63) not detectable ?
15N The additional active nucleus can be used
to label 1H atoms with the frequency of the
attached heteroatom (1H- 15N 1H- 13C) transfer
magnetization through covalents bonds using
heteronuclear J-couplings
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A 2D 1H-15N heteronuclear NMR spectrum (HSQC)
15N (ppm)
H (f1,f2)
1H (ppm)
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A 3D NMR spectrum
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A 2D plane of a 3D NMR spectrum (NOESY)
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Applications small flexible molecules that
cannot be crystallized (peptides,
oligosaccharides, ...) 3D structure
determination of proteins, nucleic acids,
protein/DNA complexes, ...) dynamics (ps to
s) electrostatics (pKa values) hydrogen
bonding (NH temperature coefficients, H2O/D2O
exchange) unfolded/partially folded states of
proteins bound solvent protein/ligand
interactions (also very weak) diffusion
coefficients analysis of biomolecules in vivo
membrane peptides and proteins (solid-state
NMR)
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Protein NMR a practical approach
Sample preparation 500 ml, 1 mM protein
solution (10 kDa ? 10 mg/ml solution ? 5 mg ?
0.5 mmol) highly efficient (gt 10 mg/l),
inducible expression system in M9 medium for
isotopic enrichment (15NH4Cl, 13C6-glucose are
expensive rich labelled media are available)
the protein must be soluble monodispersed sta
ble 20-40 C, pH 3-7 over 2-3 weeks
compatible buffers inorganic buffers
(phosphate) low ionic strength (1-100 mM)
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Spectrometers 500 MHz ? 800 MHz depending on
the size (number of residues, molecular
weight) isotopic enrichment (1H, 15N, 15N/13C,
15N/13C/2H)
Time schedule data acquisition 1-3
weeks backbone assignments 1-4 weeks side-chain
assignments 1-4 weeks list of restraint/structure
calculation 1-3 months
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Limits Molecular weight limits for protein
structure calculation (monomer) 5-15 kDa
routine 15-20 kDa usually feasible 20-30 kDa
long term project 40-50 kDa in the next
future? Molecular weight limits for
peptide/protein, protein/protein interactions (MW
of the AB complex, A lt 10 kDa) 20-30 kDa
routine 30-50 kDa feasible 50-100 kDa in the
next future
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Protein structure calculation by
NMR cloning ? expression (labeling) ? purificatio
n ? data acquisition ? sequential
assignments ? side-chain assignments ? NOE
assignments ? list of geometrical
restraints ? structure calculation ? structure
refinement ? validation ? structure/function
relationships (electrostatic potentials, surface
analysis, ligand binding sites, ...
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Protein structure calculation by NMR Structure
calculation is the determination of atoms
position (x,y,z) in a coordinate system Atom
positions in the liquid state are averaged over
space and time Structure calculation in liquids
must rely on internal coordinates in other
words, you must measure something that does not
depend on atoms position
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Protein structure calculation by NMR
HA
Geometrical restraints that allow structure
calculations distances (NOE) angles
(J-couplings)
HB
HC
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Protein structure calculation by NMR
HA
HB
HC
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Books NMR of proteins and nucleic
acids Kurt Wühtrich, Wiley (1986) NMR of
macromolecules a practical approach edited by
G. C. K. Roberts, IRL Press (1993) Protein
NMR spectroscopy principles and practice J.
Cavanagh, W. Fairbrother, A. Palmer, N.
Skelton Academic Press (1996)
NMR on the web http//www-keeler.ch.cam.ac.uk/lec
tures/
Useful links http//www.spincore.com/nmrinfo/ h
ttp//www.spectroscopynow.com/ http//www.bmrb.wi
sc.edu/
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Nuclear Magnetic Resonance in Structural
BiologyPart IThis PowerPoint presentation can
be downloaded from plaza on the ICGEB sharing
server (helix), together with a PDF copy of the
Science article ? folder NMR-1