(Bio)-applications of high-field NMR

1
(Bio)-applications of high-field NMR
2
Aims

• To give an overview of the capability of NMR to
  answer biological questions
• To make aware of limitations
• To give a basic idea about structure
  determination by NMR
• To make aware of NMR sample requirements
• To enable you to decide whether NMR would be a
  useful method in your research

3
Outline

• Introduction to biological applications of NMR
• Basics of solution structure determination of
  proteins
• Heteronuclear NMR
• NMR of nucleic acids
• NMR and dynamic phenomena
• (some more applications)

4
What can NMR do for biology ?

• 3D Structure determination of proteins and
  nucleic acids
• Assess stability and folding of proteins
• Binding studies (Proteins, DNA, Ligands)
• Protein dynamics and reactions possible to
  look at timescales between ps and days
• Elucidation of structure of biomarkers,
  metabolites, and synthetic pathways
• NMR of bio-fluids and tissues
• In vivo NMR
• Magnetic Resonance Imaging

5
3D Structure determinations
Express and purify protein (or isolate from
natural source)

• Initial characterisation
• - Identity, composition
• Concentration
• Stability (buffers, salt, pH, temperature)

GSDIIDEFGTLDDSATICRVCQKPGDLVMCNQCEFCFHLDCHLPALQDVP
GEEWSCSLCHVLPDLKEEDVDLQACKLN
Protein sequence










Acquire NMR spectra
Evaluation Sequential Assignment Extraction of
distance restraints and other structural data
3D structure
6
3D Structure determinations

• 1. The sample
• In solution
• ca. 0.2-1 mM protein solution (ca. 200-500 mL)
• Smaller than 35 kDa
• Preferentially in native form, not aggregated....
• Usually nothing paramagnetic (e.g. Cu(II), Fe(II)
  or Fe(III),
• Recombinant expression necessary for proteins gt
  8kDa (for isotopic labelling with 13C and 15N)

7
3D Structure determinations

1. The spectra

8
Fourier Transform pulse sequences

• The simplest 1D experiment

1. Radiofrequency pulse with high power
2. Recording of the free induction decay (FID)
Acquisition
Repeat - but need to make sure that excitation
from previous scan has completely vanished ?
relaxation delay
9
1D NMR
Free induction decay (FID)
Time domain
(s)
Fourier Transformation
Frequency domain
1D NMR spectrum
(s-1)
10
Typical 1H NMR spectrum of a small molecule
Recorded in 90 H2O/10 D2O
8H
aliphatic
H2O
16H (aromatic)
High field
Low field
4H
1
1
2
3
4
5
6
7
8
9
10
d 1H (ppm)
Aromatic protons are affected by electron cloud
(ring current) of aromatic ring (deshielded
the field experienced by aromatic protons is
weaker than B0, consequently the resonance
frequency is lower
11
1H NMR spectrum of a 55 amino acid protein
C225H356N70O80S9
a
NH
aliphatic side-chain
Backbone
b
CH(a)
d
Side-chain
H2O
e
NH and aromatic
0
0
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
d 1H (ppm)
12
NMR spectrum of a 66 kD proteinSize limitation

• Heavy overlap
• Broad lines

d 1H (ppm)
13
Relaxation

• Relaxation is the process that brings the excited
  system (e.g. after the rf pulse) back to its
  equilibrium state
• Transversal (T2) spin-spin
• Longitudinal (T1) spin-lattice
• Line-width of signal is reciprocally related to
  T2 fast relaxation ? broad lines
• Both T1 and T2 are dependent on molecular
  motions, e.g. for proteins molecular tumbling
  (correlation time tc (1/tumbling rate large
  molecules have long tc) and backbone
  dynamics/conformational fluctuations

14
Factors influencing the quality of NMR spectra pH

• Backbone amide protons very important for
  structure determination
• But Can dissociate and hence exchange with bulk
  protons (from water)
• Exchange leads to loss of signal intensity
• Exchange rates usually most favourable at pH 3-5

15
Factors influencing the quality of NMR spectra
ions

• salt and buffer
• proteins usually require the presence of buffers
  and/or salt
• but salt and buffer ions add to spectral noise
  ? loss of signal intensity
• Usually not more than 50-100 mM total
• NB Buffer must not contain non-exchangeable
  protons (otherwise need deuterated compound)

e.g.
O
H
O
D
C
H
C
D
2
2
C
H
C
N
H
O
H
C
D
C
N
D
O
D
2
2
2
2
C
H
C
D
2
2
O
H
O
D
Tris
Tris-d11
16
Water suppression

• Proteins are usually studied in aqueous solution
  90 H2O/10 D2O.
• D2O required for lock ensures stable field
• Typically, protein concentration a few mM
• Ca. 100 M protons from water (i.e. a 100000-fold
  excess)
• Various ways for getting rid of water signal
• Presaturation Irradiation of water resonance
  at low power before high-power rf pulse (during
  the relaxation delay)
• Watergate Selective pulse flanked by gradient
  pulses
• DPFGSE (Double Pulsed Field Gradient Spin Echo)
  or Excitation sculpting (AJ Shaka)

17
Principles of 2D NMR

• 2D NMR experiments are composed of a series of 1D
  experiments
• Involves
• Irradiation of a nucleus (as in 1D)
• Incremented delay (different for each 1D
  experiment) (also called evolution)
• Magnetisation transfer to other nucleus that is
  coupled to irradiated nucleus Signal detection
  (as in 1D)
• Results in information on correlations between
  nuclei

18
Principles of 2D NMR
1D NMR
acquisition
preparation
e.g. relaxation delay and rf pulse
t2
2D NMR are a series of 1D experiments
acquisition
evolution
mixing
preparation
t1
t2
What is detected depends on what happens during
mixing time (spin coupling)
This time period changes between the various
individual 1D experiments ? gives a second time
domain
19
Principles of 2D NMR
Generated from FID as in 1D
Repeated several hundred times with different
evolution times t1 (also called incremented delay)
1st dimension
Last FID incremented delay 0.5 s (e.g.)
time (s)
time (s)
Etc....
2nd dimension
2nd FID incremented delay 10 us
1st FID incremented delay 0
Frequency (Hz or ppm)
Fourier Transformation of the second dimension
gives the second frequency axis
20
Principles of 2D NMR Fully FT transformed
spectrum
1st dimension (F2)
(the third dimension is the peak intensity)
2nd dimension (F1)
The FID for the second dimension is generated
by the incremented delay
21
The mixing time

• Correlation between nuclei happens during the
  mixing time
• Reciprocal relationship to observed coupling
• large couplings - short mixing time
• Difficult to detect small couplings, as mixing
  takes too long, and at end of mixing time no
  magnetisation left (due to relaxation)
• If coupling through space Long range - long
  mixing time

22
Homonuclear 2D NMR

• Typical experiments
• DQF-COSY (double-quantum-filtered correlation
  spectroscopy up to 3-bond coupling
• TOCSY (total correlation spectroscopy) entire
  residues
• NOESY (nuclear Overhauser enhancement
  spectroscopy) through space
• COSY and TOCSY are based on scalar coupling
  (through bonds), NOESY on dipolar coupling

23
Identification of spin systems
E.g. Valine

• Protons have characteristic shifts
• Tabulated
• Each amino acid has a characteristic pattern in
  the various 2D spectra

0 ppm
F1
C
H
C
H
H
3
3
C
C
O
H
C
N
H
Expected TOCSY spectrum
10
10
0 ppm
F2
24
2D NMR techniques TOCSY and COSY in proteins
0 ppm
Ala10
H(b)
H(a)
O
H
TOCSY
N
C
C
H
F1
C
H
3
H(a)
H(b)
TOCSY and COSY help identifying the type of
residue
COSY
amide
10
10
0 ppm
F2
25
Regions in 2D spectra
H(b)-to-methyl (Leu,Val, Ile)
TOCSY spectrum of a decapeptide (Luteinising
hormone releasing hormone)
26
Sequential assignment
NOESY connects residues that are adjacent to each
other
0 ppm
O
O
H
H
H
N
C
C
C
C
N
H
C
H
3
C11
H
C
H
A10
F1
S
C
d
10
10
0 ppm
10
F2
27
Sequential assignment
Overlay of TOCSY with NOESY
H(a)
Amide H
28
Break
29
Recognising secondary structureChemical shift
index

• Shifts of backbone atoms are sensitive towards
  secondary structure (a helix, b sheet etc)
• Comparison of experimental shifts with tabulated
  random coil shifts (one for each amino acid)
• Quick and robust method, 95 accuracy
• Can utilise H(a) protons (13C backbone shifts
  also useful)
• Each residue with a shift larger than expected
  gets an index of 1
• Each residue with a shift smaller than expected
  gets an index of -1
• Residues within random coil shift get a 0

30
Chemical shift index Example
No recognisable secondary structure
b strands
a helix
MTKKIKCAYHLCKKDVEESKAIERMLHFMHGILSKDEPRKYCSEACAEKD
QMAHEL
-----HHHEE---------HHHHHHHHH--------------HHHHHHHH
HH----
(secondary structure prediction by jpred)
C
N
31
Secondary Structure Can also Be Characterised by
Regular Patterns of NOEs
H(a) of residue 47
NH of residue 50
NH of residue 51
Strong NOEs between NHs of adjacent residues NOE
between Ha(i) and NH(i3)
a helix
32

• Very strong sequential NOEs (from H(a) to NH of
  next residue)
• Also information on tertiary structure Strong
  NOEs between neighbouring strands

b sheet
33
Recognising the fold Analysis of backbone NOEs
Backbone trace
C
b hairpin
Residue number
a helix
N
Antiparallel b sheet
Residue number
(Predicted by homology modelling, consistent with
CSI and fold analysis)
34
Distance restraints from NOESY

• The NOE is a dipolar interaction Through space
• A cross peak between two nuclei means that
  magnetisation transfer through dipolar
  interactions between two neighbouring spins must
  have taken place during the mixing time. This
  means that the two nuclei are close together in
  space.
• The cross peak intensity is defined as follows
• I k g12g22 r-6 S J(w)

35
Real-world example 100 ms 2D NOESY of a 55 aa
protein

• 356 protons
• Ca. 2000 peaks
• Intra-residue
• Sequential
• Long-range

36
NMR restraints

• evaluated ca 1000 1H peaks
• 600 peaks unambiguously assigned
• extracted about 300 relevant distance restraints
  (3-5 Å)

37
Use of coupling constants to gain structural
information

• 3J-scalar coupling constants (extracted from
  dedicated NMR spectra) are dependent on dihedral
  (or torsional) angles

B
B

A
A
Dihedral angle
dihedron
38
Karplus relationship
Coupling constant 3J
Dihedral angle
3J a cos2 a - b cos a c a, b, c are
empirical parameters - tabulated for various
combinations of nuclei
39
Structure calculations

• A number of programs available, most popular
  XPlor, Cyana, CNS...
• Randomised starting structures
• Use distance restraints ( various other
  experimental data) together with generic atom
  masses, chirality, electric charges, Van der
  Waals radii, covalent bond lengths and angles,
  peptide geometry (constraints)
• Several methods
• 1) Distance geometry (DG) calculation of
  distance constraint matrices of for each pair of
  atoms (older method)
• 2) Restrained Molecular Dynamics (MD) Simulate
  molecular motions (e.g. torsions around bonds)
• 3) Simulated Annealing (SA) heat to a high
  temperature (e.g. 3000 K) followed by slow
  cooling steps
• Methods 2 and 3 work towards the energetically
  favourable final structure under the influence of
  a force field derived from the restraints and
  constraints

40
There is always more than one solution to the
parameter set The results of an NMR structure
determination are presented as an ensemble of
conformers
20, structures, all atoms
41
The ensemble (20 structures)
Backbone traces
42
Average structure
Ensembles are awkward to handle, if one wants to
inspect the structure, therefore calculation of
anaverage structure is useful.
Sausage Backbone representation of average
structure thickness of tube indicates deviations
between individual conformers
43
Final average structure
Initial average structure is only mean between
positions of individual atoms in different
conformers - bonds and angles strongly distorted
- need to do force-field based energy
minimisation. Newer approach Select
representative conformer
44
Validation

• Structural statistics
• Violation of restraints
• root-mean-square deviations between individual
  conformers and the mean structure
• Back-calculations does the structural model give
  rise to a NOESY spectrum that resembles the
  experimental data ?
• Is the structure physically reasonable ?
• ? Comparison of the resulting structure with
  empirical parameters
• E.g. Whatcheck and Procheck Look at bond
  lengths, angles, dihedrals, van-der-Waals
  contacts, stereochemistry.....

45
Heteronuclear NMR

• Common nuclei 15N, 13C
• Usually requires uniform labelling? expression
  of protein in cells that live on 15NH4Cl as
  single nitrogen source, and (e.g.) 13C-glucose as
  single carbon source
• Other nuclei
• 31P (the only stable isotope) useful for DNA
• 113Cd or 111Cd Cd has eight stable isotopes -
  needs enrichment

46
Labelling strategies

• Uniform
• Selective, e.g. all histidine residues
• Chain selective (for hetero-oligomers)
• Partial
• e.g. deuterate only aliphatic protons
• For solid-state NMR Use only x isotopically
  labelled nitrogen or carbon source dilute
  spins
• Or Mix uniformly-labelled with unlabelled
  protein
• Or use differentially labelled 13C sources
• Differential labelling (mixture of 2 compounds,
  observe signals of only one) Useful for
  protein/protein or protein/DNA interactions

47
15N

• Natural abundance 0.368
• Spin ½
• Receptivity relative to 1H 0.00000384
• Need isotopic labelling
• Recombinant protein expression in minimal medium
  with 15NH4Cl as single nitrogen source
• Relatively cheap ca. 15/l culture (which can be
  enough for one NMR sample)

48
1H,15N correlation (HSQC)
d 15N
105
110
115
120
125
130
135
9.0
7.0
d 1H
49
Advantages of 15N labellingQuick way to explore
folding
well folded
Unfolded/random coil
HSQC spectra taken from NMR pages of the
Max-Planck-Institut für Biochemie, Martinsried.
50
Advantages of labelled proteins
Isotope editing
15N
1H
1H
3D 1H,15N,1H HSQC-TOCSY and HSQC-NOESY
51
Advantages of labelled proteins
TOCSY NOESY
Many overlapped peaks
52
1H,1H plane from 3D 1H,15N,1H HSQC-TOCSY and
HSQC-NOESY
? Peak overlap has been remedied
53
Advantages of labelled proteinsChemical shift
perturbation(or shift mapping)

• Universally applicable to study anything that
  interacts with proteins
• small molecules (drugs, metabolites)
• other proteins
• DNA and RNA
• metal ions
• ...
• Very rapid method spectra can be recorded in few
  minutes

54
chemical shift perturbation

• Effect of copper binding
• on a 64 aa protein
• Peaks can
• Stay the same
• Shift
• Split (multiple conformers)
• (Dis)appear

G60
T9
d 15N
C12
110
E26
T31
C15
T6
E50
H61
Q51
R53
E49
120
S45
I10
E13
D47
A16
V63
T42
A14
A55
A11
I3
V41
V7
A28
130
E64
d 1H
55
Chemical shift perturbation
Weighted mean deviations
56
Triple-resonance-experiments (1H,15N and 13C)

• For facilitating sequential assignments
• Example HNCA

57
Triple-resonance-experiments
The more experiments, the less ambiguity Automated
sequential assignment possible But NMR
instrument time is precious
58
Nucleic acids NMR

• Same principles as in protein NMR

200 ms NOESY of octanucleotide d(CGCTAGCG)
O
C
N
8
H
C
N
H2 and 2
H
C
C
C
N
5
H
N
N
2
H
-

C
O
H3, 4 and 5
2
O
1
4
H
H
C
C
H1
H
H
C
C
2
3
2
-
O
Guanosine

Aromatic H8, H6, H2
http//nmr.chem.sdu.dk/dna/noesy_ba.htm
59
Nucleic acids NMR
Sequential assignment Correlation between sugar
H1 and aromatic base protons
d(CGCTAGCG)
T4
H6
T4_H1
C7
C1
G2
H1
H8
A5_H1
C3
T4_H6
G6
A5_H8
G8_H8
H1
A5
H8, 6
60
Break
61
Recent advances

• TROSY Transverse-Relaxation Optimized
  Spectroscopy enables study of larger proteins
  than previously (record so far 9 megadalton)
• Use of aligned media
• Induces dipolar coupling
• Novel sequences to measurethese residual dipolar
  couplings
• Gives information on bond orientation
• Can be used as additional information for
  structure determination
• Partial labelling

62
Example of partial labellingBacterial growth on
partially labelled 13C source

• E.g. Glycerol

O
H
O
H
C
H
C
H
2
2
C
H
O
H
1,3-13C
O
H
O
H
Castellani et al, Nature 2002.
C
H
C
H
2
2
C
H
O
H
2-13C
63
Why partial labelling ?

• Partial 13C labelling
• No scalar 13C,13C coupling
• Spectra become less crowded, can concentrate on
  dipolar couplings for structural information
• Avoid dipolar truncation effects (polarization
  transfer between two nuclei is cut off in the
  presence of a third nucleus)
• 2H reduce overlap and dipolar couplings between
  1H and 13C or 15N

64
13C distance restraints from proton-driven spin
diffusion
65
Kinetics by NMR
66
The NMR time-scale

• NMR is a relatively slow technique
• If there is more than one conformation in
  solution, two sets of peaks can be observed,
  providing the two species live for long enough
  to be detected
• Otherwise, averaging occurs
• the "NMR time-scale" for averaging of two peaks
  is the reciprocal of the difference in frequency
  of the peaks

67
Chemical exchange

• Any process in which nucleus changes between
  different environments
• E.g.
• Conformational equilibria
• Binding of small molecules to macromolecules
• Protonation/deprotonation equilibria
• Isotope exchange processes

68
Exchange regimes
Slow exchange between 2 species

• Lifetime of individual species decreases
• Exchange rate increases
• Can be achieved by raising the temperature

Intermediate Coalescence
Fast exchange
http//tesla.ccrc.uga.edu/jhp/nmr_04/notes/bcmb81
90_042604.pdf
69
H/D exchange

• Dissolve protein in 100 D2O
• Backbone amide H (and other NHx or OH groups)
  exchange with solvent deuterons.
• Exchange is fast when H is solvent exposed or in
  a flexible region (loop)
• Exchange is slow when H is buried and/or involved
  in H-bond (eg in b sheets or a helices)

70
Ligand binding studies

• With small proteins can look at protein and
  map binding site (1H,15N HSQC) via chemical shift
  changes
• With big proteins observe ligand spectrum (1H),
  check qualitatively whether ligand interacts with
  protein can do rapid screening
• Advantage Binding does not need to be strong

71
Ligand binding Transferred NOE

• Allows observation of ligand conformation bound
  to protein
• Principle Detect NOEs arising from bound state
  in unbound ligand
• Conditions
• Only works for weakly-binding ligands (ligand
  must dissociate faster than NOE decays)
• Good if protein is very big (so protein signals
  dont interfere with ligand spectrum)
• Advantage Sharp signals, as detection happens in
  the unbound form

72
Protein motions

• Not all parts of protein have same flexibility
• On 15N-labelled proteins, relaxation rates can be
  measured to derive time-scales for motion of
  whole molecule, or individual parts, e.g.
  backbone dynamics
• Can also estimate correlation time (molecular
  tumbling) and infer molecule size and shape
  (monomer/dimer, aggregation)

Residue number
Region with high flexibility
73
Metabolomics and -nomics

• Structure elucidation of novel natural compounds
• Combination of NMR with chromatography and mass
  spectrometry
• Elucidation of biochemical pathways
• protein function and mechanisms
• use of labelled precursors, e.g. 13C-labelled
  acetate, NMR analysis of products gives
  information on how metabolites are synthesised
• Metabonomics looks at complex mixtures such as
  body fluids or tissues
• With or without prior separation (chromatography)
• Analysis via comparison with known spectra
• Can be used in diagnosis of diseases

74
Various rat cells and tissues Magic-angle
spinning NMR Vast differences between tissues
http//www.bbriefings.com/pdf/855/fdd041_metabomet
rix_tech.pdf
75
MRI Magentic resonance imaging

• B0 field horizontal
• 0.5-3 Tesla
• Also uses radio-frequency pulses
• Observed nuclei are the water protons
• Contrast is achieved by different relaxation
  properties of protons in different tissues
• Gradient magnets for spatial information

MRI scanners
76
Typical images obtained by MRI
77
In vivo NMR spectroscopy (MRS)

• Diagnostic method
• Looks directly at metabolites in the body of a
  living patient (or animal)
• Examples
• 31P in muscles
• Brain diseases (Alzheimer)

78
In vivo 31P NMR of carp muscle
Normal conditions
Anoxic conditions
http//143.129.203.3/biomag/bil_bio1_spectra/bil-b
io1.html
79
In vivo NMR
Energy metabolism in microorganisms
In vivo 31P NMR spectrum of Corynebacterium
glutamicum
http//www.fz-juelich.de/ibt/genomics/coryne-phosp
horus.html
80
2D In vivo NMR of brain
Journal of NeurochemistryVolume 66 Issue 6 Page 2491  - June 1996doi10.1046/j.1471-4159.1996.66062491.x
 

A One-Dimensional (Proton and Phosphorus) and Two-Dimensional (Proton) In Vivo NMR Spectroscopic Study of Reversible Global Cerebral Ischemia
S. Brulatout, Ph. Méric, I. Loubinoux, J. Borredon, J. L. Corrèze, P. Roucher, B. Gillet, G. Bérenger, J. C. Beloeil,   B. Tiffon,   J. Mispelter, and J. Seylaz
Journal of NeurochemistryVolume 66 Issue 6 Page 2491  - June 1996doi10.1046/j.1471-4159.1996.66062491.x
 

A One-Dimensional (Proton and Phosphorus) and Two-Dimensional (Proton) In Vivo NMR Spectroscopic Study of Reversible Global Cerebral Ischemia
S. Brulatout, Ph. Méric, I. Loubinoux, J. Borredon, J. L. Corrèze, P. Roucher, B. Gillet, G. Bérenger, J. C. Beloeil,   B. Tiffon,   J. Mispelter, and J. Seylaz
Journal of NeurochemistryVolume 66 Issue 6 Page 2491  - June 1996doi10.1046/j.1471-4159.1996.66062491.x
 

A One-Dimensional (Proton and Phosphorus) and Two-Dimensional (Proton) In Vivo NMR Spectroscopic Study of Reversible Global Cerebral Ischemia
S. Brulatout, Ph. Méric, I. Loubinoux, J. Borredon, J. L. Corrèze, P. Roucher, B. Gillet, G. Bérenger, J. C. Beloeil,   B. Tiffon,   J. Mispelter, and J. Seylaz
S. Brulatout et al. J. Neurochem. 66 2491(1996).

81
Summary/Outlook

• NMR has a lot to offer for elucidating the
  structure and function of biomolecules
• Complementary method to X-ray crystallography for
  structure determination
• Can now also do membrane proteins
• Size limitation is still a problem
• Much more than a tool for structure elucidation
  (Kinetics/dynamic phenomena, biomolecular
  interactions, metabolomics and -nomics...)