NMR an introduction - PowerPoint PPT Presentation


PPT – NMR an introduction PowerPoint presentation | free to view - id: 645a7-ZDc1Z


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation

NMR an introduction


... disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in cattle ... NMR structure of the bovine prion protein. ... – PowerPoint PPT presentation

Number of Views:268
Avg rating:3.0/5.0
Slides: 86
Provided by: kul95


Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: NMR an introduction

NMRan introduction
  • by
  • Burcu Kaplan

Kurt Wüthrich
The Nobel Prize in Chemistry 2002
  • "for his development of nuclear magnetic
    resonance spectroscopy for determining the
    three-dimensional structure of biological
    macromolecules in solution"

  • Through his work at the beginning of the 1980s
    Kurt Wüthrich has made it possible to use NMR on
    proteins. He developed a general method of
    systematically assigning certain fixed points in
    the protein molecule, and also a principle for
    determining the distances between these. Using
    the distances, he was able to calculate the
    three-dimensional structure of the protein. The
    advantage of NMR is that proteins can be studied
    in solution, i.e. an environment similar to that
    in the living cell.

  • 1-the nuclear overhauser effect (NOE) as an
    experimentally accessible NMR parameter in
    proteins that can yield the information needed
    for de novo global fold determination of a
    polymer chain
  • 2-sequence-specific assignment of the many
    hundred to several thousand NMR peaks from a
  • 3-computational tools for the structural
    interpretation of the NMR data and the evaluation
    of the resulting molecular structures.
  • 4-multidimensional NMR techniques for efficient
    data collection.

Protein NMR
  • In the NMR experiments, solution conditions such
    as the temperature, pH and salt concentration can
    be adjusted so as to closely mimic a given
    physiological fluid.
  • Solutions may also be changed to quite extreme
    non-physiological conditions, for example, for
    studies of protein denaturation.
  • investigations of dynamic features of the
    molecular structures
  • structural, thermodynamic and kinetic aspects of
    interactions between proteins and other solution
    components other macromolecules or low molecular
    weight ligands

1965 postdoctoral trainingUniversity of
California, Berkeley
  • used NMR spin relaxation measurements of 17O, 2H
    and 1H in addition to EPR for studies of the
    hydration of metal ions and metal complexes

(No Transcript)
1967 Bell Telephone Laboratories
  • important qualitative NMR features of amino acids
    and proteins had already been noted and
    tentatively rationalized by 1967
  • the maintenance of what was one of the first
    super conducting high resolution NMR
    spectrometers, which operated at a proton
    resonance frequency of 220 MHz.
  • hemoproteinsblood sampled from my arm
  • with the limited sensitivity and spectral
    resolution of the instrumentation available in
    1968, the special spectral properties of
    hemoproteins were a great asset for successful
    NMR applications

1976 ETH Zürich
  • identification of the nuclear Overhauser effect
    (NOE) as a NMR parameter that can be related in
    an unambiguous way to three-dimensional
    macromolecular structures
  • NOE gives crosspeaks between resonances from
    protons close in space inter-atomic distances

  • NOE had a key role in the approach used for
    obtaining sequence-specific assignments of the
    many hundred to several thousand NMR lines in a
  • NOE are due to dipolar interactions between
    different nuclei. The intensity of the NOE is
    related to the product of the inverse sixth power
    of the internuclear distance
  • A 1 H1 H NOE is related to the through-space
    distance between a pair of atoms that are either
    not at all linked by covalent bonds
    (intermolecular NOE), or that may be far apart in
    the amino acid sequence of a polypeptide chain.

  • In 1977 the first 2D NMR spectrum of a protein
    was recorded
  • Kumar, A., Ernst, R.R., and Wüthrich, K. (1980).
    A two-dimensional nuclear Overhauser enhancement
    (2D NOE) experiment for the elucidation of
    complete proton-proton cross-relaxation networks
    in biological macromolecules. Biochem. Biophys.
    Res. Commun. 95, 16.

Contour Plot of a Proton Two-Dimensional NOESY
Spectrum of Basic Pancreatic Trypsin Inhibitor
Recorded at 360 MHz
  • 2D 1 H NMR enables the recording of selective
    interactions between pairs of hydrogen atoms, or
    groups of chemical shift-equivalent hydrogen
    atoms, without selective irradiation of
    individual resonance lines.
  • The dispersion of the resonances in a
    two-dimensional frequency plane affords greatly
    improved separation of the individual peaks.

  • four 2D NMR experiments were conducted that were
    then used for the initial protein structure
  • COSY (2D correlated spectroscopy),
  • SECSY (2D spin-echo correlated spectroscopy),
  • FOCSY (2D foldover-corrected correlated
  • NOESY (2D nuclear Overhauser enhancement

  • complete sequence-specific assignments BPTI(basic
    pancreatic trypsin inhibitor)
  • Wagner,G.and Wüthrich,K.Sequential resonance
    assignments in protein 1 H nuclear magnetic
    resonance spectrabasic pancreatic trypsin
    inhibitor.J.Mol.Biol.155 (1982) 347-366.
  • the polypeptide hormone glucagon bound to
    lipid micelles
  • Wider,G.,Lee,K.H.and Wüthrich,K.Sequential
    resonance assignments in protein 1 H nuclear
    magnetic resonance spectraglucagon bound to
    perdeuterated dodecylphosphocholine
    micelles.J.Mol.Biol.155 (1982)367-388.

Complete sequence-specific resonance assignments
for BPTI obtained using 2D NMR experiments.
Assigned residues are identified by coloured
patches covering their amide protons.(The colour
code indicates variable amide proton exchange
Kinetics of protein folding
  • Systematic investigation of hydrogen-exchange
    dynamics as a function of pH value, temperature
    and concentration of denaturants with atomic
  • K.Wüthrich,G.Wagner, NMR investigations of the
    dynamics of the aromatic amino acid residues in
    the basic pancreatic trypsin inhibitor FEBS
    Lett.1975,50 ,265 -268.
  • G.Wagner,K.Wüthrich, Correlation between the
    amide proton exchange rates and the denaturation
    temperatures in globular proteins related to the
    basic pancreatic trypsin inhibitor,J.Mol.Biol
    .1979 ,130 , 31 -37.
  • H.Roder,G.Wagner,K.Wüthrich, Amide proton
    exchange in proteins by EX1 kineticsstudies of
    the basic pancreatic trypsin inhibitor at
    variable pH and temperature,Biochemistry 1985
    ,24 ,7396 7407
  • H.Roder,G. Wagner,K.Wüthrich, Individual amide
    proton exchange rates in thermally unfolded basic
    pancreatic trypsin inhibitor,Biochemistry
    1985,24,7407 7411
  • H.Roder,K.Wüthrich Protein folding kinetics by
    combined use of rapid mixing techniques and NMR
    observation of individual amide protons,
    Proteins 1986 ,1 ,34-42.

1985 BUSI
  • first NMR structure determination of a globular
    protein, bull seminal protease inhibitor (BUSI)
  • but.....

2D 1H,1H-NOE spectroscopy (1 H,1 H-NOESY). A
stacked plot representation of a spectrum of the
small protein bullseminal proteinase
inhibitorIIA) is shown (500 MHz, 45C,
two more years of intense work....
  • Many structural constraints,mainly long-range NOE
    observations,were recorded and a mathematical
    method based on metric matrix distance geometry
    was used to calculate the three-dimensional
    structure for the protein based on these
    constraints by implementation in efficient
    software packages

1985 BUSI
Williamson,M.P.,Havel,T.F.,and Wüthrich,K.Solution
conformation of proteinase inhibitor IIA from
bull seminal plasma by 1 H nuclear magnetic
resonance and distance geometry.J.Mol.Biol.182
After BUSI
  • When I presented the structure of BUSI in some
    lectures in the spring of 1984, the reaction was
    one of disbelief and suggestions that our
    structure must have been modeled after the
    crystal structure of a homologous protein,PSTI

independently solving a new protein structure
  • amylase inhibitor Tendamistat
  • Professor Robert Huber (Nobel laureate in
    Chemistry, 1988) by X-ray crystallography
  • Kurt Wüthrich by NMR

  • Almost identical three-dimensional structures in
    terms of the global fold of the polypeptide chain
    were demonstrated in accompanying publications
  • Kline,A.D.,Braun,W.and Wüthrich,K.Studies by 1 H
    nuclear magnetic resonance and distance geometry
    of the solution conformation of the á -amylase
    inhibitor Tendamistat. J.Mol.Biol.189
  • Pflugrath,J.,Wiegand,E.Huber,R.and
    Vertesy,L.Crystal structure determination,refineme
    nt and the molecular model of the á -amylase
    inhibitor Hoe-467A.
  • J.Mol.Biol.189 (1986)383-386.

  • in the interior of the protein the two structures
    are nearly identical
  • Local differences on the surface of the protein
  • The solution structure appeared more disordered
    than the crystal structure
  • Tyrosine 14 was not observed in the X-ray

A)Family of structures of tendamistat, an
amylase inhibitor determined by NMR
spectroscopyB)ribbon diagram of the structure
with lowest energy.
  • subsequently solved NMR structure of rabbit
    metallothionein was completely different from an
    independently solved rat metallothionein crystal
  • different polypeptide folds
  • different coordinating ligands to metals
  • Nature rejected NMR structure article

  • crystal structure of rat metallothionein was
    re-determined and the correct crystal structure
    was found to be identical with the NMR structure
    of the rabbit, rat and human metallothioneins
    that Wüthrich solved from 1985 to 1990
  • it took six years before the crystal structure
    was redetermined and found to coincide with the
    NMR structure!

  • 3 D 4 D NMR
  • Isotope labelling of the molecules with the
    NMR-active nuclei 15 N and 13 C led to the
    development of heteronuclear three-dimensional
  • Heteronuclear relaxation provides the basis for
    NMR studies of molecular dynamics in a
    macromolecule,showing that the parts of a
    molecule that appear disordered in a structure
    determination are often associated with high
  • Protein folding

Size limitation ?
  • Recently reported structures represent molecular
    weights up to the order of megaDa in extreme
    cases.Compared to these large single-crystal
    structures,NMR solution structures generally
    concern smaller molecules, typically below 30
    kDa,and they are often less precise.
  • With TROSY and CRINEPT techniques it is now
    possible to assign resonances and study a protein
    assembly as large as 900 kDa,as shown in the
    recent study of the molecular chaperone
    GroEL-GroES complex
  • Pervushin,K.,Riek,R.,Wider,G.and
    Wüthrich,K.Attenuated T 2 relaxation by mutual
    cancellation of dipole-dipole coupling and
    chemical shift anisotropy indicates an avenue to
    NMR structures of very large biological
    macromolecules in solution.Proc.Natl.Acad.
    Sci.USA 94 (1997)12366-12371.
  • Riek,R.,Wider,G.,Pervushin,K.and
    Wüthrich,K.Polarization transfer by
    cross-correlated relaxation in solution NMR with
    very large molecules.Proc.Natl.Acad.Sci.USA 96
  • Fiaux,J.,Bertelsen,E.B.,Horwich,A.L.and
    Wüthrich,K.NMR analysis of a 900K GroEL-GroES
    complex.Nature 418 (2002)207-211.

  • Complete cancellation of transverse relaxation
  • for amide sites in a very large protein,in which
    all hydrogen atoms but the exchangeable ones such
    as the amide hydrogen have been replaced with
    deuterium,the effect of cross-correlated
    relaxation of different relaxation mechanisms
    such as NH dipole -dipole relaxation and N
    chemical shift anisotropy leads to enhanced
    relaxation for the downfield component of the H N
    doublet,while for the upfield component,the two
    relaxation processes can mutually cancel.Since
    the chemical shift anisotropy is field dependent
    and the dipole dipole relaxation is field
    independent for large molecules,the two effects
    will mutually cancel at a magnetic field of
    approximately 950 MHz

  • 20 of the 14 000 structures
  • Wüthrichs group contributed with more than 50

Prion Protein
  • PrPc benign cellular form
  • predominantly expressed in neuronal tissue
  • transmissible spongiform encephalopathies (TSEs)
    are a group of fatal neurodegenerative diseases,
    which include Creutzfeldt-Jakob disease (CJD) in
    humans and bovine spongiform encephalopathy (BSE)
    in cattle

Prion protein
  • A structure determination for the C-terminal half
    of the mouse prion protein in April 1996, barely
    10 days after the BSE-crisis in Great Britain
    broke into the open.
  • In 1997 characterization the structure of the
    intact prion protein showing that the N-terminal
    half of the molecule forms a highly flexible,
    extended "tail". The prion protein thus presented
    a striking illustration of the unique power of
    NMR to characterize partially structured
    polypeptide chains.
  • the three-dimensional structure of the benign
    cellular form (PrP C ) includes a flexibly
    disordered 100-residue tail linked to the
    N-terminal end of a globular domain .

Partially folded polypeptide chains
  • difficult to crystallize
  • the chain segments that are disordered in
    solution will either be ordered by intermolecular
    contacts in the crystal lattice, or they will not
    be visible by diffraction methods.

NMR structure of the recombinant murine prion
First the well-ordered structure of a fragment
comprising the C-terminal residues 121-231 was
determined.Then the intact protein 23-231 was
studied and it was found that the N-terminal
23-126 segment formed an extended,highly flexible
coil with high mobility.
NMR structure of the bovine prion protein. In
the C-terminal globular domain of residues
126230, -helices are green, an antiparallel
sheet is blue, and non-regular secondary
structure is yellow the unstructured
N-terminal tail of residues 23125 is white.
  • static picture of the unstructured chain
  • additional NMR experiments can provide
    information on the frequencies of the rate
    processes that mediate transitions between
    discrete states of the molecule within the
    conformation space spanned by the static bundle
    of NMR-conformers
  • Wüthrich, K. (1995) Acta Cryst. D 51,
    249270. NMRthis other method for protein and
    nucleic acid structure determination.

Visual impression of the variation of the bovine
prion protein structure during a time period of
about 1 nanosecond. The superposition of 20
snapshots illustrates that the globular domain
maintains its mean geometry, whereas the tail
undergoes large-scale changes with time.
  • Considering that the mechanism of transformation
    of PrP C into the aggregated, disease-related
    form, PrPSc, of mammalian prion proteins is
    still subject to speculation, the observation of
    this flexible tail has been highly intriguing.

  • Riek, R., Hornemann, S., Wider, G., Billeter, M.,
    Glockshuber,R., and Wüthrich, K. (1996). NMR
    structure of the mouse prion protein domain
    PrP(121231). Nature 382, 180182.
  • Riek,R.,Hornemann,S.,Wider,G.,Glockshuber,R.and
    Wüthrich,K. (1997) NMR characterization of the
    full-length recombinant murine prion protein
    mPrP(23-231).FEBS Lett.413 282-288.
  • Zahn, R., Liu, A., Lührs, T., Riek, R., von
    Schroetter,C., Lopez Garcia, F. et al. (2000).
    NMR solution structure of the human prion
    protein. Proc. Natl Acad. Sci. USA, 97, 145150.
  • Lopez Garcia, F., Zahn, R., Riek, R. and
    Wüthrich, K. (2000) NMR structure of the bovine
    prion protein Proc. Natl. Acad. Sci. USA 97,

Thorsten Lührs, Roland Riek, Peter Güntert and
Kurt Wüthrich
NMR Structure of the Human Doppel Protein
J. Mol. Biol. (2003) 326, 15491557
Prion Protein
  • PrPc knockout mice is not susceptible to prion
  • Reintroduction restores susceptibility
  • Büeler, H., Aguzzi, A., Sailer, A., Greiner, R.
    A.,Autenried, P., Aguet, M. et al. (1993). Mice
    devoid of PrP are resistant to scrapie. Cell, 73,
  • Fischer, M., Rülicke, T., Raeber, A., Sailer, A.,
    Moser, M., Oesch, B. et al. (1996). Prion protein
    (PrP) with amino-proximal deletions restoring
    susceptibility of PrP knockout mice to scrapie.
    EMBO J. 15, 12551264.
  • Flechsig, E., Shmerling, D., Hegyi, I., Raeber,
    A. J., Fischer, M., Cozzio, A. et al. (2000).
    Prion protein devoid of the octapeptide repeat
    region restores susceptibility to scrapie in PrP
    knockout mice. Neuron,27, 399408. USA, 98,

Doppel Protein
  • Knockout strains with no resistance
  • Knockout strains developing signs of ataxia
    within 70 weeks after birth
  • discovery of a novel gene locus (Prnd) 16 kb
    downstream of Prnp and its product, the doppel
    protein (Dpl).
  • Moore, R. C., Lee, I. Y., Silverman, G. L.,
    Harrison, P. M., Strome, R., Heinrich,C. et al.
    (1999). Ataxia in prion protein (PrP)-deficient
    mice is associated with upregulation of the novel
    PrP-like protein doppel.
  • J. Mol. Biol. 292, 797817.

Dpl disease?
  • An involvement of Dpl in prion diseases or a role
    in neural differentiation is unlikely.
  • Behrens, A., Brandner, S., Genoud, N.
    Aguzzi, A. (2001). Normal neurogenesis and
    scrapie pathogenesis in neural grafts lacking the
    prion protein homologue Doppel. EMBO Rep. 2,
  • Thus two distinct neurological diseases,
    simultaneously with overexpression of two
    distinct proteins, seem to be cured by benign
    cellular form of prion protein.

  • sequence identity between Dpl and PrP is only
    about 20, so that experimental determination of
    Dpl 3D-structure is a significant addition to the
    data available as a foundation for functional
    studies of these proteins.

  • PCR from Prnd, Dpl coding region
  • Cloning in E.coli pRSETA
  • 17 residue N-terminal histidine tail
  • Engineered thrombin cleavage site
  • DNA sequencing
  • N-terminal Edman sequencing
  • MALDI-TOF mass spectrometry

  • Expression
  • incubation of soluble protein fraction with
    Ni-NTA agarose
  • Refolding of protein using a linear gradient of
    0100 (v/v) of buffer B (100 mM NaPi, 10 mM Tris
    (pH 8.0), 10 mM imidazole).
  • The refolded hDpl(24152) was then eluted with
    buffer B containing 500 mM imidazole.

  • N-terminal histidine tail cleavage
  • Separation of cleavage products by
    cation-exchange chromatography, CM52-cellulose
  • Dialysis
  • Lyophilized/ fresh solution purified protein

NMR spectroscopy
  • 1.3 mM solution of uniformly 13 C 15 N-labelled
    protein in 95 (v/v) H2O,
  • 5 (v/v) 2H2O
  • for sequence-specific polypeptide backbone
    assignments TOCSY
  • for the collection of conformational
    constraints,three 3D NOESY-experiments
  • 1.9 mM solution of unlabeled protein in 100 2H2O
  • for 2D NOESY

NOESY ( 750 MHz)
  • 3D 15N/13C-resolved 1H,1H-NOESY H2O
  • 3D 13C-resolved 1H,1H-NOESY in D2O
  • 3D 13C-resolved 1H,1H-NOESY in D2O
  • 2D 1H,1H-NOESY in D2O

15N,1H-correlated spectroscopy (COSY)
  • Resonance doubling for some residues
  • SDS-PAGE MALDI-TOF 95 homogenous
  • Oxidation states of the 4 Cysteine residues
  • 2 disulfide bridges
  • Resonance doublings seen for protein fragment
    hDpl(52152), globular domain

Chemical shift assignments
  • incomplete assignments
  • backbone amide protons of Y91, K143, C145 and
  • all side-chain protons of R32, K34 and K143
  • H? of R27,
  • H? 1 of H31
  • H? of P86
  • H? of D87
  • H? of I89
  • H? of C145
  • H? of F147
  • Proline trans conformation 13 C? 32 ppm

  • For doubled peaks separated by less than 0.02 ppm
    in the 1 H-dimension and/or less than 0.1 ppm in
    the 15 N or 13 C dimension of the
    heteronuclear-resolved 3D 1H,1H-NOESY spectra
    ,only one chemical shift in each dimension was
    assigned and the sum of the peak intensities was

(No Transcript)
  • 2. For doubled resonances separated by more
    than these limits, the more intense peak was
    arbitrarily added to the input, with an intensity
    corresponding to the sum of the intensities of
    the two peaks.

  • Computational...
  • Peak lists of the four NOESY spectra were
    generated by interactive peak picking with the
    program XEASY and automatic integration of the
    peak volumes with the program SPSCAN
  • Automated combined NOE cross peak assignment and
    three-dimensional protein structure determination
    was obtained using the programs CANDID and DYANA

Input for the structure calculation of hDpl(24
152) and characterization of the energy-minimized
NMR structure of the globular domain 52152
3-D structure human doppel protein,
(No Transcript)
3-D structure human doppel protein,
Beta sheet
  • anti-parallel ?-sheet comprising residues 5860
    (?1) and 8890 (?2)
  • was identified on the basis of strong H ?
    (F59)H?(I89), H? (G88) HN (I89) and H? (A58)
    HN(F59) NOEs
  • with the inter-strand hydrogen bonds HN
    (H90)O(A58) and HN (I60) O(G88) implicated
    by the atom coordinates of the energy-refined

Bundle of 20 energy-minimized conformers with the
lowest DYANA target functions of the polypeptide
segment 52149 of hDpl(24152) obtained by
super-position of the N, C? and C atoms of the
residues 5290, 101121 and 126141 for best fit.
The backbone (cyan), and the two disulfide
bridges (yellow) are displayed.
Packing of amino acid side-chains in the core of
hDpl(24152).The residues shown originate from
loops preceding and following helix a1 (magenta),
helix a1 (green), helix a3 (orange) and backbone
is shown in cyan.
Environment of the residue F59, shown in green,
formed by residues in the helices ?2 and ?3
(orange), and in the first ? -strand and the
preceding loop (magenta).
disulfide bond in C-terminal
hDpl(24152) (red) vs mDpl(26155) (white)
Regular secondary structure elements are
indicated next to the ribbons, the chain ends are
indicated by sequence numbers. The disulfide
bridge homologous to that in the prion protein is
shown in yellow ,rmsd 1.8 A
Tentative alignment
  • CirclesN-glycosylation sites
  • Dots conserverd a.a.
  • Hyphen deletions
  • pairwise amino acid identities between hDpl/mDpl,
    hDpl/hPrP and mDpl/hPrP are 79, 20 and 20,

hDpl vs mDpl
  • 22 Amino acid substitutions
  • 9 on the surface of the molecule none appear to
    be involved in long-range or medium-range
    interactions with other segments of the
    polypeptide chain, so that they are unlikely to
    contribute significantly to structural
    differences between the two proteins.

helix ?2 b and the loop connecting ?2b with ?3
nine differences G118A, Q121S, K122R, P123E,
D124K, N125Q and K126D, and a dipeptide insertion
following position 126 in the hDpl amino acid
sequence. Interestingly, although four variations
of the amino acid sequence near helix ?2 b
involve charged residues, the net local charge of
the segment 117127 (hDpl numeration) is
invariant between the two proteins.
Core substitutions
  • I109V and V136I strictly preserve the hydrophobic
    core packing
  • F104L and L139A could relate to the structural
    differences between the two proteins at the start
    of helix ? 2a

hDpl (red) vs hPrP (white)
rmsd 2.5 A
Conserved residues
  • I68 (I139 in hPrP), F70 (F141), Y77 (Y149) and
    Y78 (Y150)
  • G71 (G142)
  • N81 (N153)
  • P86(P158)
  • Y91 (Y163)
  • F104 (F175), V105 (V176) and C108 (C179)

  • The side-chain of T112 (T183) in ?2 forms a
    long-range hydrogen bond to the ?-sheet
  • in hDpl this interaction involves the hydroxyl
    proton of T112 and O of G57 on strand ?1
  • in hPrP it involves the hydroxyl oxygen atom of
    T183 and HN of Y162 on strand ?2.
  • This switch in donor/acceptor function is
    reflected in largely different ?1 angles of this
    Thr residue, which is virtually
    solvent-inaccessible in both hDpl and hPrP.
  • In hPrP, this core hydrogen bond has been shown
    to contribute significantly to the thermodynamic
    stability of PrP, and it probably has a similar
    role in Dpl.

A cartoon of glycosylated,GPI-anchored hPrP and
hDpl. ? -helices are red and yellow, ? -strands
are cyan, and the segments with non-regular
secondary structure within the C-terminal domain
are gray. The GPI anchor and the glycan moieties
are black, and the disulfide bridges are green.
conserved glycosylation site
significant differences
  • the ?1-strand and the sequentially adjacent
    residues show a displacement of corresponding
    C?atom coordinates of approximately 8 A . The
    ?-sheet is shifted by two residues towards helix
    ?1, resulting in a significant rearrangement of
    the loop connecting ?1 and ?1 with respect to
    helix ?3, while leaving the position of the loop
    connecting ?1 and ?2 virtually unaffected.
  • different packing of the side-chains in this
    molecular region, where residue F59 of the
    ?1-strand in hDpl is part of the hydrophobic core
  • The C-terminal polypeptide segments of hPrP and
    hDpl have a low level of sequence conservation
    and different 3D structures. While in hPrP the
    helix ?3 proceeds almost to the C terminus, the
    helix ?3 of Dpl terminates after C140 in the
    common disulfide bond.
  • The peptide segment between the end of ?3 and the
    chain end of hDpl has a non-regular secondary
    structure and is folded against the loop
    connecting ?2 with ?2,giving the hDpl molecule an
    overall more contracted appearance when compared
    with hPrP.
  • the additional disulfide bond C94C145 in hDpl
    would be sterically incompatible with a regular
    a-helix structure beyond about residue 142.

hydrophobic cleft
Negative charges are shown in red, positive
charges in blue, and residues belonging to the
hydrophobic core and other hydrophobic
side-chains are shown in yellow.The charged
residues are identified by the one-lettersymbols
and the sequence numbers.
  • In the pathology of TSEs the refolding of PrPc
    to PrPsc appears to be a central event.
  • Substitutions of the residues D178, V180, T183,
    R208, V210, E211 and Q212 in hPrP, which
    correspond to G107,I109, T112, R134, V136, Q137
    and E138 in hDpl have been identified as genetic
    mutations that relate to increased probability to
    develop familial forms of TSE.
  • Three of these residues are conserved in the
    sequences of hDpl and hPrP, i.e. T112 (T183),
    R134 (R208) and V136 (V210), and are found in
    corresponding locations in the 3D structure

Taken from Kurt Wüthrich, NMR solution
structure of the human prion protein PNAS ,
2000, 97(1), 145150
  • A Nobel laureate ?
  • No critique about the techniques used !
  • Maybe the fact that there was incomplete
    assignments too much simplification of NOESY
  • But results, I believe , will not much
    contribute to prion disease research directly
  • Further functional studies with hDpl,can be a
    good feedback to determine the function of hPrP
  • One should be a genius or crazy to try to deal
    with such a complex data generated by NMR!
  • Still....

GOD BLESS Kurt Wüthrich
  • Harald Schwalbe, Kurt Wüthrich,the ETH
    Zürich,and the Development of NMR Spectroscopy
    for the Investigation of Structure,Dynamics,and
    Folding of Proteins ChemBioChem 2003,4,135 142
  • Karin Markides, Astrid Gräslund , Mass
    spectrometry (MS)and nuclear magnetic resonance
    (NMR) applied to biological macromolecules
    Advanced information on the Nobel Prize in
    Chemistry 2002,9 October 2002
  • Kurt Wüthrich, NMR studies of structure and
    function of biological macromolecules , Nobel
    Lecture, December 8, 2002
  • Arthur G. Palmer and Dinshaw J. Patel , Kurt
    Wüthrich and NMR of Biological Macromolecules,
    Structure, Vol. 10, 16031604, 2002
  • Kurt Wüthrich, The way to NMR structures of
    proteins , Nature structural biology, 8(11)
    923-925 2001
  • Kurt Wüthrich, The second decade - into the
    third Millenium, Nature structural biology,NMR
    supplement ,july 1998

  • Thorsten Lührs, Roland Riek, Peter Güntert and
    Kurt Wüthrich, NMR Structure of the Human Doppel
    Protein, J. Mol. Biol. 2003,326, 15491557
  • Huaping Mo, Richard C. Moore , Fred E. Cohen ,
    David Westaway , Stanley B. Prusiner , Peter E.
    Wright and H. Jane Dyson, Two different
    neurodegenerative diseases caused by proteins
    with similar structures PNAS, 2001 98(5),
  • Ralph Zahn, Aizhuo Liu, Thorsten Lührs, Roland
    Riek, Christine von Schroetter, Francisco Lopez
    Garcia, Martin Billeter ,Luigi Calzolai, Gerhard
    Wider, and Kurt Wüthrich, NMR solution structure
    of the human prion protein PNAS , 2000, 97(1),
  • Francisco Lopez Garcia, Ralph Zahn, Roland Riek,
    and Kurt Wü thrich, NMR structure of the bovine
    prion protein PNAS 2000 97 (15) 83348339

  • http//www.nobel.se/chemistry/laureates/2002/wuthr
  • http//www.mol.biol.ethz.ch/wuthrich
  • http//www.scripps.edu/mb/wuthrich

About PowerShow.com