NMR an introduction

1
NMRan introduction

• by
• Burcu Kaplan

2
Kurt Wüthrich
3
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"

4
NOBEL

• 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.

5
NOVEL

• 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
  protein
• 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.

6
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

7
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

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9
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

10
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

11
NOE

• 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
  protein.
• 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.

12
2D NMR

• 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.

13
Contour Plot of a Proton Two-Dimensional NOESY
Spectrum of Basic Pancreatic Trypsin Inhibitor
Recorded at 360 MHz
14
2D NMR

• 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.

15
1980

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

16
1982

• 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.

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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
rates
18
Kinetics of protein folding

• Systematic investigation of hydrogen-exchange
  dynamics as a function of pH value, temperature
  and concentration of denaturants with atomic
  resolution.
• 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.

19
1985 BUSI

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

20
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,
H2O-solution).
21
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

22
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
(1985)295-315.
23
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

24
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

25
Suprise...

• 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
  (1986)377-382.
• 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.

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Tendamistat

• 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
  diffraction.

27
A)Family of structures of tendamistat, an
amylase inhibitor determined by NMR
spectroscopyB)ribbon diagram of the structure
with lowest energy.
28
1985

• subsequently solved NMR structure of rabbit
  metallothionein was completely different from an
  independently solved rat metallothionein crystal
  structure
• different polypeptide folds
• different coordinating ligands to metals
• Nature rejected NMR structure article

29
1992

• 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!

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today

• 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
  NMR
• 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
  mobility.
• Protein folding

31
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
  (1999)4918-4923.
• Fiaux,J.,Bertelsen,E.B.,Horwich,A.L.and
  Wüthrich,K.NMR analysis of a 900K GroEL-GroES
  complex.Nature 418 (2002)207-211.

32
TROSY

• Complete cancellation of transverse relaxation
  effects
• 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

33
NMR in PDB

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

34
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

35
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 .

36
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.

37
NMR structure of the recombinant murine prion
protein
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.
38
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.
39
NMR

• static picture of the unstructured chain
  segments
• 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.

40
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.
41
PRION

• 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.

42
Prion

• 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,
  83348399

43
Thorsten Lührs, Roland Riek, Peter Güntert and
Kurt Wüthrich
NMR Structure of the Human Doppel Protein
J. Mol. Biol. (2003) 326, 15491557
44
Prion Protein

• PrPc knockout mice is not susceptible to prion
  infection
• 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,
  13391347.
• 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,
  23522357.

45
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.

46
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,
  347352.
• Thus two distinct neurological diseases,
  simultaneously with overexpression of two
  distinct proteins, seem to be cured by benign
  cellular form of prion protein.

47
Rationale

• 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.

48
MM

• 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

49
MM

• 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.

50
MM

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

51
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

52
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

53
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

54
Chemical shift assignments

• incomplete assignments
• backbone amide protons of Y91, K143, C145 and
  F147,
• 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

55
CSA

• 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
  used.

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57
CSA

• 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.

58
CSA

• 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

59
Input for the structure calculation of hDpl(24
152) and characterization of the energy-minimized
NMR structure of the globular domain 52152
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3-D structure human doppel protein,
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3-D structure human doppel protein,
63
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
  structure.

64
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.
65
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.
66
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).
67
C-terminal
68
disulfide bond in C-terminal
69
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
70
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,
  respectively

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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.

72
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.
73
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

74
hDpl (red) vs hPrP (white)
rmsd 2.5 A
75
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)

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H-bond

• 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.

77
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
78
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.
•  

79
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.
80
TSE

• 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

81
hPrP
Taken from Kurt Wüthrich, NMR solution
structure of the human prion protein PNAS ,
2000, 97(1), 145150
82
Critique?

• A Nobel laureate ?
• No critique about the techniques used !
• Maybe the fact that there was incomplete
  assignments too much simplification of NOESY
  spectrum
• 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....

83
GOD BLESS Kurt Wüthrich
84
REFERENCES

• 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

85
References

• 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),
  23522357
• 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),
  145150
• Francisco Lopez Garcia, Ralph Zahn, Roland Riek,
  and Kurt Wü thrich, NMR structure of the bovine
  prion protein PNAS 2000 97 (15) 83348339

86
Web...

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

87
THANKSFOR YOUR PATIENCE