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Nuclear Magnetic Resonance in Structural BiologyNuclear Magnetic Resonance in Structural Biology
Part IPart I
• • the physical principlethe physical principle
•• the spectrometerthe spectrometer
•• the NMR spectrumthe NMR spectrum
•• the applications (what you can do)the applications (what you can do)
•• the samplethe sample
•• the limits (what you cannot do)the limits (what you cannot do)
• • protein structure calculationprotein structure calculation
• • referencesreferences
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Nuclear Magnetic Resonance in Structural BiologyNuclear Magnetic Resonance in Structural Biology
Part II Part II
Journal ClubJournal 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 principleThe physical principle
B (magnetic field)
Energy
nuclear spin, I=½
+½
-½
= B/2
At B=11.7 T (tesla, 104 Gauss) the resonant frequency for 1H is 500 MHz
(earth magnetic field = 30-60 T; magnetic stirrer = 0.1 T)
In NMR, excitation is achieved through short (s) electromagnetic (radio-frequency) pulses.
The E is very small (10-2 cal) low population difference low sensitivity
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The spectrometerThe spectrometer
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The NMR spectrumThe 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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The chemical shiftThe chemical shift
The chemical shift depends on:
• the atom type (NH, aliphatic CH, aromatic CH, ...)
• the amino acid type (Ala, Phe, ...)
• the chemical (spatial) environment
The resonant frequency of a certain atom is called chemical shift.
For convenience, the chemical shift is expressed as follows:
(sample) – (reference)
spectrometer frequency(ppm) = 106 •
Advantages:
• more compact annotations
• independent on the spectrometer field
In practice, the 1H chemical shifts are in the range 0-10 ppm
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The 1D spectrum of a peptideThe 1D spectrum of a peptide
N
H
C
H
C N
O HCH2 8 7 6 5 4 3 2 ppm
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The assignment problemThe assignment problem
Which resonance corresponds to which atom?
N
H
C
H
C N
O H
CH3
CH
H3Cthrough-bonds magnetization transfer (J-couplings)
through-space magnetization transfer (NOE)
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The 2D spectrumThe 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.
f1
f2
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The 2D spectrum of a peptide (DQF-COSY)The 2D spectrum of a peptide (DQF-COSY)
Ac-GRGGFGGRG-NHAc-GRGGFGGRG-NH22
N
H
C
H
C N
O HCH2
Ph
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The 2D spectrum of a peptide (TOCSY)The 2D spectrum of a peptide (TOCSY)
Ac-GAc-GRRGGFGGGGFGGRRG-NHG-NH22
N
H
C
H
C N
O H
CH2
CH2
CH2
~
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The 2D spectrum of a peptide (ROESY) The 2D spectrum of a peptide (ROESY)
Ac-GRGGFGGRG-NHAc-GRGGFGGRG-NH22
+
N
H
C
H
C N
O
H
CH2
~
+
++
+ + + ++
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The NMR experiment as a black box ?The NMR experiment as a black box ?
sample NMR spectrum?
Playing around with r.f. pulses
(and magnetization):
• power
• length (s)
• phase (x, -x, y, -y)
• receiver phase (x, -x, y, -y)
• time delays
p1 p2 p3 detection (s)
t1t2
exp-1 exp-2 exp-3 ....
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The 2D spectrum of a protein (NOESY)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 NMRFrom 1D to 2D and 3D NMR
nuclear magnetic resonance in structural biology, part Inuclear magnetic resonance 1D
in structural biology, part I
nuclear magnetic resonance 2D
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The NMR spectrumThe 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 A 2D 11H-H-1515N heteronuclear NMR spectrum (HSQC)N heteronuclear NMR spectrum (HSQC)
15N
1H
C
H
1H (ppm)
15N (ppm)
H (f1,f2)
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A 3D NMR spectrumA 3D NMR spectrum
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A 2D plane of a 3D NMR spectrum (NOESY)A 2D plane of a 3D NMR spectrum (NOESY)
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Applications 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 approachProtein NMR: a practical approach
Sample preparationSample preparation
• 500 l, ~1 mM protein solution (10 kDa 10 mg/ml solution 5 mg 0.5 mol)
• highly efficient (> 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:solublemonodispersedstable 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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Time scheduleTime 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
SpectrometersSpectrometers
500 MHz 800 MHz depending on :
the size (number of residues, molecular weight)
isotopic enrichment (1H, 15N, 15N/13C, 15N/13C/2H)
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LimitsLimits
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 < 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 NMRProtein structure calculation by NMR
cloning
expression (labeling)
purification
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 NMRProtein 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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HB HC
HA
Geometrical restraints that allow structure calculations:
• distances (NOE)
• angles (J-couplings)
Protein structure calculation by NMRProtein structure calculation by NMR
HB HC
HA
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HB HC
HA
Protein structure calculation by NMRProtein structure calculation by NMR
HBHC
HA
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Books:Books:
• NMR of proteins and nucleic acidsKurt Wühtrich, Wiley (1986)
• NMR of macromolecules: a practical approachedited by G. C. K. Roberts, IRL Press (1993)
• Protein NMR spectroscopy: principles and practiceJ. Cavanagh, W. Fairbrother, A. Palmer, N. SkeltonAcademic Press (1996)
NMR on the web:NMR on the web:
http://www-keeler.ch.cam.ac.uk/lectures/
Useful links:Useful links:
http://www.spincore.com/nmrinfo/
http://www.spectroscopynow.com/
http://www.bmrb.wisc.edu/
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Nuclear Magnetic Resonance in Structural BiologyNuclear Magnetic Resonance in Structural BiologyPart IPart I
This 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