Applications of high-field NMR

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					(Bio)-applications of high-
        field NMR




                              1
                    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


                                                 2
                 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)

                                            3
 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                   4
                 3D Structure determinations

 GSDIIDEFGTLDDSATICRVCQKPG       Express and              Initial characterisation
 DLVMCNQCEFCFHLDCHLPALQD
 VPGEEWSCSLCHVLPDLKEEDVDL
 QACKLN
                                 purify protein           - Identity, composition
                                 (or isolate from         - Concentration
                                 natural source)          - Stability (buffers, salt,
 Protein sequence                                            pH, temperature)




Acquire NMR spectra
                             Evaluation:
                                                               3D structure
                             Sequential Assignment
                             Extraction of distance restraints
                             and other structural data                              5
 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
      > 8kDa (for isotopic labelling with 13C and 15N)



                                                      6
3D Structure determinations




     2. The spectra




                              7
Fourier Transform pulse sequences
• The simplest 1D experiment:
 1. Radiofrequency pulse with high power




                                                   2. Recording of the free induction decay (FID)
               0.10
               0.10   0.20
                      0.20   0.30
                             0.30   0.40
                                    0.40   0.50
                                           0.50   0.60
                                                  0.60   0.70
                                                         0.70



              Acquisition



Repeat - but need to make sure that
excitation from previous scan has
completely vanished  relaxation delay



                                                                                            8
                              1D NMR

   “Free induction
   decay” (FID)
                     0.10   0.20    0.30   0.40   0.50   0.60   0.70   Time domain
                                                                        (s)


                                   Fourier Transformation




                                                                       Frequency domain
1D NMR spectrum


                                                                               (s-1)


                                                                                          9
     Typical 1H NMR spectrum of a small
                  molecule
                                              Recorded in 90% H2O/10% D2O


                                                    8H



                                                     aliphatic
               16H (aromatic)       H2O
 Low                                                                  High
 field                                                    4H          field


10       9    8       7         6     5       4      3           2   1
                                                                     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                        10
1H   NMR spectrum of a 55 amino acid protein
                               C225H356N70O80S9
 NH         a
            H    O                                     aliphatic side-chain
 H                    Backbone
      N     C    C
            CH2 b
                                       CH(a)
      N              Hd
                      Side-chain
                NH
      H e                              H2O

      NH and aromatic




      10         9        8   7    6        5      4      3    2     1    0
                                                                          11
                                       d   1H(ppm)
 NMR spectrum of a 66 kD protein:
        Size limitation



- Heavy overlap
- Broad lines




  10   9    8     7   6     5      4   3   2   1   0
                          d 1H (ppm)               12
                  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
                                                 13
  fluctuations
   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

                                         14
   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.:
                     OH                          OD
              H2C                          D2C
   HO   CH2    C          NH2   DO   CD2    C         ND2
              H2C                          D2C
                     OH                          OD
              Tris                                          15
                                     Tris-d11
              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) 16   or
    “Excitation sculpting” (AJ Shaka)
         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                             17
              Principles of 2D NMR
    1D NMR:
                                     acquisition
                preparation
              e.g. relaxation
                                          t2
              delay and rf
              pulse


       2D NMR are a series of 1D experiments:
                                                       acquisition
         preparation    evolution        mixing
                                t1                         t2

                                                   What is detected depends
This time period changes between the
                                                   on what happens during
various individual 1D experiments
                                                   mixing time (spin coupling)
 gives a second time domain                                                18
                               Principles of 2D NMR
                           Generated from FID as in 1D
                                                                 Repeated several hundred
 1st dimension:                                                  times with different
                                                                 evolution times t1 (also
                                0.10
                                0.10    0.20
                                        0.20      0.30
                                                  0.30    0.40
                                                          0.40   called incremented delay)
                                                                   0.50
                                                                   0.50   0.60
                                                                          0.60  0.70
                                                                                0.70


                                                                  Last FID; incremented delay =
                                       time (s)
                                                                  0.5 s (e.g.)
          2nd dimension:




                                                     time (s)

                                                                    Etc....




                                                                 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
                                                                                             19
          Principles of 2D NMR: Fully FT
              transformed spectrum

                                               1st dimension (F2)




                                                (the third dimension is the
                                                peak intensity)

2nd dimension (F1)
                                                                       20
The “FID” for the second dimension is generated by the “incremented delay”
          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                            21
      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
                                                     22
        Identification of spin systems
                                    E.g. Valine:
• Protons have          0 ppm
  characteristic shifts
• Tabulated
• Each amino acid has
  a characteristic
  “pattern” in the
  various 2D spectra        F1

      H 3C       CH 3
             H
             C

             C          O
             H   C
        N
        H
                             10   Expected TOCSY spectrum
                            10          F2                    ppm
                                                            0 23
     2D NMR techniques: TOCSY and
           COSY in proteins
                      0 ppm
Ala10                                                H(b)
        H(a)
          H      O
                                     TOCSY
    N     C      C
    H
          C H3            F1
                                         H(a)
              H(b)

TOCSY and COSY help
identifying the type of                           COSY
residue
                                     amide
                           10                                24
                                10           F2             0 ppm
Regions in 2D spectra

                NH-to-sidechain crosspeaks
                aromatic

                H(a)-to-H(b)
                H(a)-to methyl
                (Ala, Thr,Leu,
                Val, Ile)
                H(b)-to-methyl (Leu,Val,
                Ile)




                TOCSY spectrum of a
                decapeptide (Luteinising
                hormone releasing
                hormone)              25
                     Sequential assignment
             NOESY connects residues that are adjacent to each other


                                    0 ppm

             O                  O
H     H                H
N     C      C   N     C        C
                 H
      C H3                      C11
A10                  H C H
          COSY                         F1
          TOCSY        S
                           Cd
 Intra-residue Cys11                                       NOESY
 Intra-residue Ala10
 Inter-residue,
                      10
 sequential          10
                                                                        26
                                                                       0 ppm
                                                      F2
       Overlay of TOCSY with NOESY
                          Sequential
                          assignment
H(a)




                                     27
             Amide H
Break




        28
   Recognising secondary structure:
        Chemical 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      29
               Chemical shift index: Example
  No recognisable                       b strands
  secondary structure



                                                        a helix




MTKKIKCAYHLCKKDVEESKAIERMLHFMHGILSKDEPRKYCSEACAEKDQMAHEL
-----HHHEE---------HHHHHHHHH--------------HHHHHHHHHH----
 (secondary structure prediction by jpred)
                                                    C



                                                              30
                                                         N
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
NH’s of adjacent
residues
NOE between Ha(i) and
NH(i+3)
                                   a helix           31
          • Very strong
            sequential NOEs
            (from H(a) to NH of
            next residue)
          • Also information on
            tertiary structure:
            Strong NOEs
            between
            neighbouring strands




b sheet                      32
                          Recognising the fold: Analysis of
                                 backbone NOEs
                                                      Backbone trace
                 50



                 40                                     C
Residue number




                                                                             b hairpin
                 30



                 20

                                                       a helix                         N
                 10



                  0                                                       Antiparallel
                      0   10      20   30   40
                               Residue number
                                                 50
                                                                          b sheet

                                                      (Predicted by homology modelling,
                                                      consistent with CSI and fold
                                                      analysis)
                                                                                  33
    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)




                                                        34
Real-world example: 100 ms 2D NOESY of a 55 aa protein

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




                                                   35
NMR restraints
                  evaluated ca 1000
                 1H peaks




                 600 peaks
                 unambiguously
                 assigned

                  extracted about
                 300 relevant
                 distance restraints
                 (3-5 Å)




                              36
         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

             B
                                        Dihedral angle
                 A
                         dihedron
                     X                                       37
         X
                                Karplus relationship
Coupling constant 3J




                                            Dihedral angle
                       3J = a cos2 a - b cos a + c;
                       a, b, c are empirical parameters - tabulated for
                                                                          38
                       various combinations of nuclei
               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
                                                                 39
  the restraints and constraints
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”




                                                 40
                  20, structures, all atoms
The ensemble (20 structures)




    Backbone traces            41
              Average structure
Ensembles are awkward to handle, if one
wants to inspect the structure,
therefore calculation of an
average structure is useful.



“Sausage”: Backbone
representation of
average structure;
thickness of tube
indicates deviations
between individual
conformers
                                          42
            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          43
                  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.....                   44
             Heteronuclear NMR
• Common nuclei: 15N, 13C

• Usually requires uniform labelling
   expression of protein in cells that live on
  15NH Cl as single nitrogen source, and (e.g.)
       4
  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                                45
              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              46
                      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)
                                               47
        1H,15N     correlation (HSQC)

d 15N
            H      O
                                        105
        N   C      C
        H                               110
            C H3
                                        115

                                        120

                                        125

                                        130

                                        135
             9.0
                       d 1H       7.0     48
             Advantages of 15N labelling:
             Quick way to explore folding




                  well folded                         Unfolded/random coil
                                                                                 49
HSQC spectra taken from NMR pages of the Max-Planck-Institut für Biochemie, Martinsried.
  Advantages of labelled proteins
               Isotope editing

    15N




   1H




          1H


3D [1H,15N,1H] HSQC-TOCSY and HSQC-NOESY
                                       50
               Advantages of labelled proteins

TOCSY
NOESY




                                                 51
        Many overlapped peaks
1H,1H   plane from 3D [1H,15N,1H] HSQC-TOCSY and HSQC-NOESY




                                                              52
                    Peak overlap has been remedied
    Advantages of labelled proteins:
      Chemical 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
                                           53
                chemical shift perturbation

d 15N    Effect of copper binding
         on a 64 aa protein
                                      T9                    G60

         Peaks can
         -Stay the same
         -Shift                                         C12
110
         -Split (multiple conformers)
         -(Dis)appear
                            T31                                    E26
          T6                            C15
                      E50
                          R53                               Q51                H61
120                     E49
        E13                    S45                           I10
                            D47
                                        V63                              A16
               T42                A14
                                                      A55
                 I3                             A11
130     V41             A28       V7

                                              E64

                                                                                     54
                                         d    1H
       Chemical shift perturbation
 5


4.5


 4
                                      1
                   Δδ  δ 1 H        δ 15 N
                                 2             2
3.5
                                      7
 3
                            Weighted mean deviations
2.5


 2


1.5


 1


0.5


 0
           4
           6
           8


           2


           6
           8
           0
           2
           4


           8
           0




           6


           0




                                                       8


                                                       2
                                                       4
         12




         52
           6
 T2

   4
 T6
         P8

          0




          0




          2
          4




          4
                                                      6
         24




         48




                                                     60
       A1
       A1
       A1


       A2


       E2
       A2
       A3
       V3
       V3


       S3
       K4




       A4


       E5




                                                   S5


                                                   E6
                                                   E6
       L3
 Q




        I1




       T2




       T4
       T4




       T5

                                                    I5
       C




       D
       Q




       G




                                                   G
                                                       55
     Triple-resonance-experiments
             (1H,15N and 13C)
• For facilitating sequential assignments
• Example: HNCA




                                            56
Triple-resonance-experiments




            The more experiments, the less
            ambiguity
            Automated sequential assignment
            possible                               57
            But: NMR instrument time is precious
                                   Nucleic acids NMR
  • Same principles as in                                      200 ms NOESY of octanucleotide
    protein NMR                                                        d(CGCTAGCG)
                               O

                     N         C
          8                C       NH
                HC                              H2’ and 2”
                           C       C
                     N
          5’                   N        NH2
-O        CH2
                O                             H3’, 4’ and 5’    {
4’   HC              CH   1’
           CH       CH2                             H1’
     3’                   2’
           O-


          Guanosine



                                                                    {
                                                               Aromatic
                                                               H8, H6, H2                58
http://nmr.chem.sdu.dk/dna/noesy_ba.htm
                   Nucleic acids NMR
Sequential assignment:
Correlation between sugar H1’ and aromatic base protons

                                     d(CGCTAGCG)


  T4
        H6                  T4_H1’
                                                              C7
                                                      C1
             H1’     H8               G2
                            A5_H1’                    C3
                                                 G6        T4_H6
                                 A5_H8
                                           G8_H8
                     H1’
   A5

                                                             59
                                         H8, 6
Break




        60
               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 measure
    these residual dipolar couplings
  – Gives information on bond
    orientation
  – Can be used as additional
    information for structure
    determination
• Partial labelling
                                              61
          Example of partial labelling:
Bacterial growth on partially labelled 13C source
• E.g. Glycerol:
        OH        OH
    H2C           CH2
             CH
             OH
[1,3-13C]



      OH         OH           Castellani et al, Nature 2002.
   H2C           CH2
            CH
            OH                                           62
                   [2-13C]
          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
                                                63
13C   distance restraints from proton-
          driven spin diffusion




                                     64
Kinetics by NMR




                  65
          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


                                             66
          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


                                           67
                                                       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
                                                                                  68
http://tesla.ccrc.uga.edu/~jhp/nmr_04/notes/bcmb8190_042604.pdf
              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)



                                               69
        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
                                           70
  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
    don’t interfere with ligand spectrum)
• Advantage: Sharp signals, as detection
  happens in the unbound form

                                                      71
            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
                                                           72
       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
                                                              73
Various rat cells
and tissues
Magic-angle
spinning NMR

Vast differences
between tissues




http://www.bbriefings.com
/pdf/855/fdd041_metabom
                     74
etrix_tech.pdf
   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
MRI scanners         different relaxation
                     properties of protons in
                     different tissues
                   • Gradient magnets for
                     spatial information
                                         75
Typical images obtained by MRI




                                 76
  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)

                                         77
        In vivo 31P NMR of carp muscle


       Normal conditions




       Anoxic conditions




                                                             78
http://143.129.203.3/biomag/bil_bio1_spectra/bil-bio1.html
                          In vivo NMR




               Energy metabolism in microorganisms
     In vivo 31P NMR spectrum of Corynebacterium glutamicum

http://www.fz-juelich.de/ibt/genomics/coryne-phosphorus.html   79
           I
           n

2D In vivo NMR of brain
            V
            i
           v
           o

           N
           M
           R

           S
           p
           e
           c
           t
           r
           o
           s
           c
           o
           p
           i
           c

           S
           t
           u
           d
           y              S. Brulatout et al.
                          J. Neurochem. 66
           o                              80
           f
                          2491(1996).
           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...)
                                            81
                                          81

				
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