introduction to nmr spectroscopydl.icdst.org/pdfs/files/a7f0ba581eace744a5d739a6f0a2e5db.pdfnmr...
TRANSCRIPT
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Introduction to NMR spectroscopy
Swiss Institute of Bioinformatics
I.Phan & J. Kopp
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NMR: the background
Complex technique. Requires knowledge in: Mathematics
Physics
Chemistry
Biology
(Medicin)
Involves a lot of computing
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N.M.R.
Nuclear Magnetic Resonance spectroscopy
imaging
solid-state
....and much more
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structure
imaging
dynamics
foldingkinetics
in-vivo
NMR applications
binding
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Can NMR be used for:
Medical diagnosis?
Drug-design?
Computing?
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NUCLEAR Magnetic Resonance
NMR can detect atoms with a nuclear spin 1/2
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Some nuclei with spin 1/2
10031P
10019F
0.3715N
1.1113C
99.981H
Natural abundance
(%)
Isotope12C and 16O do not have a spin 2H, 14N have spin 1
Is this good news forsolving the structureof proteins by NMR?
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NUCLEAR Magnetic Resonance
Can NMR be used to detect: Ozone?
Salt?
Butter?
A brain tumor?
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Nuclear MAGNETIC Resonance
Nuclei with spin ½ behave like a magnet
Placed in a constant magnetic field, they will align with that field
H HB
How do we create a constant magnetic field?
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NMR instrumentation
The spectrometer
super-conductingmagnet
RF field generator
sample tube goes in here
NOTE:the proteinis in solution[1mM in water]
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NMR is not sensitive
Signals from the nuclei are measured in parts per million [ppm] of the static field strength
NMR experiments require: concentrated samples
strong fields = (very) big magnets
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Protein structure file in the Protein Data Bank
Structure data in the Protein Data Bank * X-ray crystallography : over 80% * NMR : about 16%
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The cost of NMR
Superconducting magnet : no resistance, no current loss
Requires cooling to almost absolute zero Liquid helium (+ Liquid nitrogen for insulation)
Strong magnetic field Requires special infrastructure
Minimize field disturbance
NMR is a very expensive technique!
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superconducting magnet
radio frequency unit
A typical NMR lab
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Nuclear Magnetic RESONANCE
H
H
H
HH
H
H
H
RF field
wavetofrequencytransformation
sound
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Nuclear Magnetic RESONANCE
H
H
H
HH
H
H
H
RF field
wavetofrequencytransformation
sound
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In an NMR experiment, the energy input to make the nuclei resonate is produced by a Radio Frequency (RF) PULSE
RF field: magnetic field B1 perpendicular to the constant
magnetic field B0
time
currentin coil
t
pulse
IProtein sample
Apply and detect a current in the coil
Constant magnetic field B0
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The PULSErotates the bulk magnetisation M0 around x-axis
The angle of rotation is proportional to the duration of the RF pulse time t
B0
What is the effect of the pulse?
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NMR experiments
Depending on the length of the pulses and delay between pulses, different effects are measured Variety of NMR experiments: variety of spectra
Depending on the frequency of the RF pulse, different nuclei can be detected Proton 1H – NMR 15N – NMR spectra 13C – NMR spectra ...etc...
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How is an NMR signal detected?
After the pulse, the nuclei return to their ground energy state
The nuclei precess back to their start position
Precessing induces a current that is detected by a coil in the NMR spectrometer
As the nuclei return to equilibrium, the induced current decreases back to zero = Free Induction Decay (FID)
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time
I
The NMR signal: a Free Induction Decay (FID)
Current induced by one precessing spin decays after RF pulse
The FID signal
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Nuclear Magnetic RESONANCE
One spin: one bar magnet
Many spins: bulk magnetisation
Depending on the length of the RF pulse, the bulk magnetisation of an ensemble of spins will flip at a different angle with respect to the static field (B
0)
After the pulse, each spin precesses individually and gives rise to an FID
?
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FID for an ensemble of spins
Current induced by precessing spins decays after RF pulse
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Nuclear Magnetic RESONANCE
H
H
H
HH
H
H
H
RF field
wavetofrequencytransformation
sound
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NMR signal processing
NMR spectrum: a superposition of signals
One signal: FID of one nucleus
Interpretation is made easier by a simple mathematical formula that transforms of the FID from the time domain to the frequency domain
Fourier transformation
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I FT
resonancefrequency
FT
Fourier Transformation
frequencytime
time
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Richard Ernst (ETHZ)
Nobel 1991
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NMR structures
How do we get a protein 3D structure with NMR?
NMR experiments
Data collection
Spectrum assignment
Structure calculation
Method assessment: is NMR really worth the effort?
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NMR experiments
1D NMR 'hit, measure' (1D pulse sequence)
RF
t1
FT
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1H NMR experiment
1D NMR 1 peak for each proton in a distinct environment within
the protein
height ∝ number of structurally identical H ( -CH3)
position (shift) ∝ electronegativity of surrounding Minute differences in shifts: measured in Part Per Million of the field
width ∝ protein size size expressed in terms of tumbling (correlation) time -> there is an experimental limit to the size of the proteins one can
determine by NMR ! we need tricks, more tricks....
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1H NMR spectrum of ethanol
Electronegative group: proton signal is shifted compared to the reference
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1H NMR shifts in different molecular environment
What does it mean for NMR spectra of: lipids? Sugars? DNA? Proteins?
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1H NMR spectra of proteins
Problem Number 1: overlap. The larger the protein, the more protons, the worse the overlap
Solutions:- higher field, stronger magnet?- 2D experiments- 15N, 13C labelling
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1H NMR spectra of proteins
Problem number 2: in NMR terms, a large protein [ > 250 residues] means also besides overlapping peaks:
faster decaying FID
broader lines
poorer sensitivity
No practical solution for this problem (?).
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NMR experiments
2D NMR 'hit, wait, hit, measure' (2D pulse sequence)
RF
t1
FTRF
t2
A slice from above gives a map of crosspeaks.
crosspeaks
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The diagonal contains the 1-dimensional spectrum
Off diagonal cross peaks contain new information
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NMR experiments
Useful 2D NMR techniques H-COSY: through-bond connections visible if protons are at most 3
bonds apart
H-NOESY: through-space connections visible if d < 6Å
C
H
C
H
C
H C
H
peak on 2D map withposition f(H),f(H)
d
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Heteronuclear experiments
When overlap is too bad for solving a structure by 1H NMR alone
Label protein with 15N and 13C Expensive, time-consuming, bad yields
...but 15N and 13C resonate at completely different frequencies from 1H
Multi-dimensional experiments ... and much more...
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Heteronuclear experiments
15N-1H COSY
15N
1H
N
H
C
H
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NMR assignment
Assignment of spectra method developped by K. Wuethrich
first protein 3D-structure solved by NMR in 1983
many protons, even in small proteins complex problem relatively simple solution arguably the most fastidious stage of protein structure
determination by NMR
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Kurt Wuethrich (ETHZ)
Nobel 2002
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NMR assignment
Assignment of spectra (K. Wuethrich) map individual amino acids using COSY spectrum
set of 2D peaks particular for each side-chain 'spin-system', or relative arrangement of protons
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COSY amino acid patterns
C
H
C
H
peak on 2D map withposition f(H),f(H)
Do you see potentialproblems for proteins?
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Degeneracy of COSY patterns
Some amino acids sharethe same spin system
... for DNA it's even worse!
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NMR assignment
Assignment of spectra (K. Wuethrich) map individual amino acids using COSY spectrum
set of 2D peaks particular for each side-chain 'spin-system', or relative arrangement of protons
locate individual amino acids within the sequence using NOESY spectrum (sequential assignment)
through-space connections from HA(i) to HN(i+1)
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NOESY sequential assignmentNOESY gives through-space connections:peak on 2D map with position f(H),f(H) if distance d < 6Å
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More from the NOESY spectrum
Secondary structure patterns
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Nuclear Overhauser Effect
Through-space connections, as given by the NOESY experiment, are the key to solving a protein structure by NMR
Long-range interactions give the fold of the protein chain
C
H C
H
d
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adjacent beta-strands
Positioning of two helices relative to each other
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NMR assignment
Assignment of spectra (K. Wuethrich) map individual amino acids using COSY spectrum
set of 2D peaks particular for each side-chain 'spin-system', or relative arrangement of protons
locate individual amino acids within the sequence using NOESY spectrum (sequential assignment)
through-space connections from HA(i) to HN(i+1)
compile list of all other peaks arising from through-space connections (NOEs)
-> set of pairwise distance restraints
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NMR distance restraints
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NMR structure calculation
How to get from the NOEs to the 3D model?
pairwise distances
atomic coordinates
?
d1
d2
d3
(x,y,z) (x,y,z)
(x,y,z)
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NMR structure calculation
Distance geometry solves the triangular inequality
Simulated annealing (Michael Nilges) fancy monte-carlo simulation ("travelling salesman
problem")
Torsion angle dynamics (Peter Güntert) hybrid method in dihedral angle space
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Michael Nilges (EMBL)
XPLOR structure calculation and molecular dynamics algorithm
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Peter Guentert (ETHZ)
DYANA structure calculation programme with integrated automatic assignment protocol
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NMR structures
What you see...
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NMR structures
...is not what you get from NMR!
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NMR structures
NMR-derived distance restraints (NOEs) are upper-limits ("d < 6 Å")
transformation of distances to coordinates gives many solutions
NMR relies on cooperativity of distance restraints: the more restraints per residue, the better defined the
structure
one NOE set produces a family of structures: loops: few experimental restraints -> bad definition -> "fuzzy" core: lots of long-range restraints -> good definition -> "compact"
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NMR structure determination
Difficulties protein in solution: protein has to be soluble
insensitive method: requires high concentrations of proteins
overlap: direct determination of 3D structures for small proteins only (150-200 residues)
Advantages no chemical modification necessary
protein in solution: no crystal packing artefacts, allows direct binding experiments, hydrodynamic and folding studies
assignment of labile regions possible: no gaps in structure
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Detecting unstructured loops
NMR spectrum spectrum shows no long-distance interactions but
sequential assignment is possible
backbone is free to adopt a range of conformations: greater variation in structure coordinates for loop residues
Crystallography electron density map shows nothing at all
structures will have gaps for residues in mobile loops
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Prion protein by NMR
Not explorableby XRay
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Ribosome structure by XRay
Impossible taskfor NMR
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NMR applications
SAR: "shot-gun" approach to drug design
Exploring Fibrils by solid-state NMR
Protein folding mechanism
NMR of proteins in bicelles (semi-crystalline state)
TROESY
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Structure Activity Relationship by NMR
Drug design method developed by Abbott Laboratories
Aim: discover high-affinity ligands for proteins
Example application: antiviral agent against the human papillomavirus
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Quantum computing by NMR
Computers molecules
Information atomic nuclei state
Programming radio-frequency pulse
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Quantum computing by NMR
from bits: 0 1 to qubits: |0,0> |0,1> |1,0> |1,1>
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Quantum computing by NMR
WHY? atoms change energy states very quickly
-- much more quickly than even the fastest computer processors.
each qubit can take the place of an entire processor -- 1,000 ions of barium could take the place of a 1,000 processor
computer.
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Quantum computing by NMR
WHAT sort of problem a quantum computer would be able to solve in principle?
Large-scale cryptography
modelling and indexing very large databases
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Quantum computing by NMR
WHO wants to build a quantum computer? IBM/MIT/Berkeley/ Stanford
Isaac Chuang (IBM) Neil Gershenfeld (MIT) “Enabling technology” for NMR-based quantum computing; scale up
to 10-40 qubits
Harvard/ MIT/Los Alamos David Cory (Harvard) Quantum algorithms and NMR-based systems
Oxford University David Deutsch, Jonathan Jones Ion-trap and NMR implementations; quantum information theory
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finally...the R.Ernst view of NMR
mathematics
physics
chemistry
biology
medicin
N
M
R