Prsentation PowerPoint

1
Insight into the conformational behaviour of
unfolded proteins from residual dipolar
couplings Martin Blackledge, Phineus Markwick,
Malene Ringkjobing Jensen, Dominique Marion,
Laurence Blanchard, Klaartje Houben Institut de
Biologie Structurale CEA-CNRS-UJF Grenoble,
France Pau Bernado Parc Cientifico, Barcelona,
Spain Stephan Grzesiek, Sebastian
Meier Biozentrum Basel, Switzerland Marcus
Zweckstetter, Marco Mukrasch, Christian
Griesinger MPI, Göttingen, Germany
2
Unfolded Proteins The Dark Side of Structural
Biology

• Natively disordered regions are present in at
  least 30 of proteins coded in eukaryotic genome
  sequences.
• Implicated in transcriptional regulation,
  translation and cellular signal transduction,
  molecular recognition, protein phosphorylation .
  
• as well as neurodegenerative pathology
• A description of the conformational behaviour of
  natively disordered and denatured proteins is
  central for understanding protein stability and
  folding, misfolding and induced folding upon
  binding
• How do we deal with the structure/function
  paradigm.with no structure?

Uversky Prot. Sci 2002, 11,739 Dunker et al
Biochemistry 2002, 41 6573
3
Conformational Behaviour of Unstructured Proteins
A Challenge for Structural Biology

• Molecular Description
• Single structure is no longer appropriate
• An ensemble of interchanging structures is
  required to fully describe conformational
  behaviour
• Paradigm - Identification of rules of
  conformational sampling

4
NMR of Unfolded Proteins
Time and ensemble averaged conformational
properties of the unfolded chain
5
NMR of Unfolded Proteins
Time and ensemble averaged conformational
properties of the unfolded chain
Shortle Ackerman, (2001), Science 293, 487-489,
Mohana-Borges et al (2004) J. Mol. Biol. 34,
1131-114, Meier et al (2004) J. Mol. Biol. 344,
1051...
6
Weak Alignment
Dipolar coupling
B0
?
S
ris
I
Dipole-dipole interaction between two magnetic
moments
Incomplete orientational sampling - residual
dipolar coupling
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Structural Information from Residual Dipolar
Couplings
Azz
Alignment frame (tensor) defined by 5 independent
parameters - (Aa,Ar,a,b,g)
Residual dipolar couplings report on the
orientation of internuclear vectors relative to
molecular alignment tensor
Axx
Ayy
8
RDCs in Unfolded Proteins
B0
Averaged dipolar couplings values depend on
average of bond vector orientation relative to B0
field
9
RDCs in Unfolded Proteins
?
P2(cos ?)
?
Averaged dipolar couplings values depend on
average of bond vector orientation relative to B0
field
10
Averaged values are dependent on average of bond
vector orientation relative to B0 field
How can we describe this restricted integral over
angular space analytically?
11
Averaged values are dependent on average of bond
vector orientation relative to B0 field
N-n
i
How can we describe this restricted integral over
angular space analytically? Annila et al have
used a random flight chain model Disordered
homopolymer is treated as a Gaussian chain
composed of N statistical segments of given
persistence length
hz
n
L
Louhivouri et al. J.Am.Chem. Soc.
2003,125,15647. Obolensky, Schlepckow, Schwalbe,
Solov'yov, J. Biomol NMR In Press
12
RDCs in Unfolded Proteins in Partially Aligned
Systems
RDCs are not averaged to zero in aligned random
flight polymer chains
13
RDCs in Unfolded Proteins in Partially Aligned
Systems

• For unfolded proteins (complex heteropolymers)
  conformational properties depend on amino-acid
  type
• Analytical predictions are not yet feasible for
  such complex systems
• Can we use statistical sampling to predict RDCs
  in unfolded states?

14
Conformational Sampling of Unstructured Proteins
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
K
R
D
E
P
.
?
.
?
15
Conformational Sampling of Unstructured Proteins
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
K
R
D
E
P
.
?
.
?
16
Conformational Sampling of Unstructured Proteins
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
K
R
D
E
P
.
?
.
?
17
Conformational Sampling of Unstructured Proteins
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
K
R
D
E
P
.
?
.
?
18
Conformational Sampling of Unstructured Proteins
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
19
Conformational Sampling of Unstructured Proteins
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
K
R
D
E
P
.
?
.
?
20
Conformational Sampling of Unstructured Proteins
Calculate associated RDCs
Calculate alignment of each conformer
Zweckstetter Bax J.A.C.S. 2000, 122, 3791
21
Conformational Sampling of Unstructured Proteins
Bernado, Blanchard, Timmins, Marion, Ruigrok
Blackledge Proc.Natl.Acad.Sci. 102, 17002 (2005)
22
Calculation of average RDCs for conformational
ensemble (50,000)
23
A Database of Amino-Acid Specific Conformations
500 X-ray single chain structures Resolution lt
1.8 Å and B lt 30 Å2
Secondary structural elements removed
Lovell et al. Proteins 2003, 50,437.
24
Conformational Sampling of Unstructured Proteins
Steric exclusion is introduced on a residue
specific basis
Levitt (1976) J. Mol. Biol. 104, 59-107.
25
RNA polymerase (PL) from Sendai virus
Nucleocapsid (NRNA)
PX
RNA
PMD
NCORE
NTAIL
N
PX
PMD
P
474
1
320
568
PCT
PNT
Phosphoprotein P positions L onto the
RNA-nucleoprotein complex
Tarbouriech et al. Nat. Struct. Biol. 7 (2000)
777-81
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Phosphoprotein X-domain PXTwo domains, one
folded and one unfolded
568
?3
His-tag
474
27
Conformational behaviour of the natively unfolded
domain of protein X from Sendai Virus
RDCs measured in PX aligned in 5 C12E6/hexanol
Folded region
28
Conformational Sampling of Unstructured Domain of
Protein X
.
ltDijgt
Calculation of average RDCs for conformational
ensemble (50,000)
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Conformational Sampling of Unstructured Domain of
Protein X
ltDN-NHgt
calc
exp
Random ?/? sampling Simple non-bonding exclusion
30
Conformational Sampling of Unstructured Domain of
Protein X
Sampling ?/? using amino acid specific
propensities Simple non-bonding exclusion
Pro
Leu
Ala

31
Conformational Sampling of Unstructured Domain of
Protein X
Protein X from Sendai virus - containing both
folded and unfolded domains
Model quantitatively reproduces distribution and
proportion of RDCs in folded and unfolded domains
Bernado, Blanchard, Timmins, Marion, Ruigrok
Blackledge Proc.Natl.Acad.Sci. 102, 17002 (2005)
32
Spectrosopic and scattering data reproduced by
the ?/? propensity conformational model
Bernado, Blanchard, Timmins, Marion, Ruigrok
Blackledge Proc.Natl.Acad.Sci. 102, 17002 (2005)
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How general is this model?
NH RDCs measured in Staphyloccocal Nuclease ?131?
mutant in Urea
Shortle Ackerman Science 2001, 293, 487.
NH RDCs measured in urea-denatured apo-myoglobin
Mohana-Borges et al JMB 2004, 340, 1131.
NH RDCs measured in urea-denatured Ubiquitin
Meier Grzesiek
34
Detection of local and long-range order in
natively unfolded proteins ?-synuclein Tau K18
Collaboration with M.Zweckstetter, C.Griesinger
and coll. MPI Göttingen
35
Conformational Behaviour of ?-Synuclein

• ?-Synuclein is a pre-synaptic protein abundant
  in protein-rich aggregates found in human
  brain affected by Parkinsons disease
• Compactness linked to inhibition of fibrillation
  - burial of aggregation nucleation site

36
Conformational Behaviour of ?-Synuclein
Simulated using Flexible-meccano
Experimental
Aligned in lyotropic phase
Aligned in Bacteriophage
RDCs measured in ?-Synuclein
Additional fine structure due to local/long-range
contacts?
Bertoncini, Jung, Fernadez, Hoyer, Griesinger,
Jovin, Zweckstetter P.N.A.S. 102, 1430, (2005)
37
Effects of Long-Range Contacts on Predicted RDCs
Contacts dij lt 15Å between ?C atoms present in
different 20 residue segments
Bernado, Bertoncini, Griesinger, Zweckstetter
Blackledge J.A.C.S. 127, 17968, (2005)
38
Long range order and short range disorder in
?-Synuclein
Contact between N and C terminal domains
reproduces RDC data Terminal domains have
complementary charges - interaction may be
electrostatic RDC fine structure disappears in
presence of polyamines and Urea
Bernado, Bertoncini, Griesinger, Zweckstetter
Blackledge J.A.C.S. 127, 17968, (2005)
39
Conformational Behaviour of ???????????

• Natively unstructured protein regulating the
  organization of neuronal microtubules
• Found in high concentrations in neurofibrillary
  tangles of Alzheimers disease
• Mechanism of transition from normal to
  pathological form remains poorly understood

40
Conformational Behaviour of ??????????? from RDCs
RDCs measured in 4-repeat domain (K18) in
uncharged polyacrylamide gel
-DNH
DLKNV
SVQIVYKPVDLSNV
SVQIVYKPVDLSKV
DFKDR
243
372
Good agreement with statistical simulation with
the exception of 4 homologous regions in each
domain - RDC inversion local turn conformation?
41
Conformational Behaviour of ??????????? from RDCs
AA-specific propensity based sampling
Molecular Dynamic sampling
?
?
?
Incorporation of this information into
statistical sampling approach Replace
amino-acid specific backbone dihedral angle
distributions by conformations extracted from MD
Simulation
42
Conformational Behaviour of ??????????? from RDCs
-DNH
LDFKDR
PDLKNV
LDLSNV
VDLSKV
243
372
Turns identified in R2 and R3 intercalated
between regions with ?-sheet propensities
Mukrasch, Markwick, Biernat, von Bergen, Bernado,
Griesinger, Mandelkow, Zweckstetter, Blackledge 
J.Am.Chem.Soc. 129, 5235-5243 (2007).
43
Conformational Behaviour of Urea-Unfolded Proteins
Chemical denaturants stabilize the unfolded with
respect to the folded state and therefore provide
valuable insight into the protein folding
process The mechanism of protein unfolding due
to the presence of chemical denaturants is not
well understood Can RDCs help?
Collaboration with S.Meier, S.Grzesiek and coll.
Biozentrum, Basel
44
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Measurement of fixed geometry dipolar couplings
NH-N, ?C-?H, ?C-??
H?
HN
HN
H?
H?
HN
HN
H?
H?
HN
HN
45
Conformational Behaviour of Urea-Unfolded
Ubiquitin
NNH, ?C?H RDCs measured in 8M urea-denatured
Ubiquitin at pH 2
Independent scaling
46
Averaged values are dependent on average of bond
vector orientation relative to B0 field
How can we describe this restricted integral over
angular space?
P2(cos ?)
?
47
RDCs in Unfolded Proteins
?
48
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Angular averaging from simulations of unfolded
Poly-Alanine ensembles
P2(cos ?)
1.0
0.5
0.0
-0.5
?
49
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Angular averaging from simulations of unfolded
Poly-Alanine ensembles
Effective angular averaging upon increasing
levels of positive ?
Angular averaging of dipolar couplings in the
peptide unit depends on both local geometry and
extendedness of the chain
50
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Comparison of calculated and experimental
couplings NNH, ?C?H
Standard coil
51
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Comparison of calculated and experimental
couplings NNH, ?C?H, NHNH (i1), NHNH(i2),
NH?H(i-1)
More Extended Database
52
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Measurement of interproton dipolar couplings
NHNH (i1), NHNH(i2), NH?H , NH?H(i-1)
H?
HN
HN
H?
H?
HN
HN
H?
H?
HN
HN
7 RDCs per peptide
53
Mapping the Conformational Landscape of
Urea-Unfolded Ubiquitin using Residual Dipolar
Couplings
All RDCs scaled using optimal scaling for
H?(i-1)HN
Comparison of experimental and simulated 1H-1H
RDCs using standard coil sampling conditions for
urea-unfolded ubiquitin
54
Mapping the Conformational Landscape of
Urea-Unfolded Ubiquitin using Residual Dipolar
Couplings
Increasing the level of extendedness of the chain
provides a more consistent reproduction of all
RDCs
Meier, Grzesiek, Blackledge  J.Am.Chem.Soc. 129,
ASAP (2007).
55
Increasing the level of extendedness of the chain
provides a more consistent reproduction of all
RDCs
Extended conformers
?-extended conformation
Polyproline II conformation
56
Conformational Behaviour of Urea-Unfolded
Ubiquitin
?
3J scalar couplings indicate extension is general
and not localised in PPII
Peti W, Henning M, Smith LJ, Schwalbe H (2000) J
Am Chem Soc 12212017-12018
57
Mapping the Conformational Landscape of
Urea-Unfolded Ubiquitin using Residual Dipolar
Couplings
?
?
Comparison of calculated and experimental
RDCs for optimal sampling conditions of
urea-unfolded ubiquitin Identifies sampling of
more extended ??? conformations than are present
in the statistical coil database
Meier, Grzesiek, Blackledge  J.Am.Chem.Soc. 129,
ASAP (2007).
58
Mapping the Conformational Landscape of
Urea-Unfolded Ubiquitin using Residual Dipolar
Couplings
Extended sampling
Coil database
Meier, Grzesiek, Blackledge  J.Am.Chem.Soc. 129,
ASAP (2007).
59
Mapping the Conformational Landscape of
Urea-Unfolded Ubiquitin using Residual Dipolar
Couplings
60
Mapping the Conformational Landscape of
Urea-Unfolded Ubiquitin using Residual Dipolar
Couplings

• Measurement of multiple RDCs, in combination with
  statistical coil sampling provides a detailed
  description of the conformational behaviour of
  the unfolded state
• Amino acids in Urea-unfolded Ubiquitin sample
  residue-specific ?/? basins, but are pushed to
  more extended conformations in presence of
  denaturant
• We are able to develop a precise, self consistent
  picture of conformational behaviour of the
  denatured protein, in agreement with RDCs, scalar
  couplings and chemical shifts, in addition to
  available biophysical data reporting on long
  range behaviour (SAXS, PFG, FRET)

61
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62
Conformational Behaviour of ??????????? from RDCs
?
50,000 strong ensemble for each ?/? segment
Dependence of RDC profiles with respect to the
backbone conformations of S316
63
Predicting Local Conformational Behaviour in Tau
Accelerated Molecular dynamics simulations of
pentadecapeptides centred on amino acids K254,
S285, S316 and K347 in explicit solvent
(AMBER8) Acceleration is achieved by enhancing
probability of transition between low energy
states. Efficient method for sampling free-energy
surface
64
Standard database
Extended database
Comparison of calculated and experimental
couplings from protein X for different levels of
chain extension
65
Dr. Malene Ringkjobing Jensen, IBS Dr. Phineus
Markwick, Institut Pasteur, Paris Dr. Pau
Bernado, Parc Cientific, Barcelona, Spain Prof.
Stephan Grzesiek, Dr. Sebastian Meier, Biozentrum
Basel, Switzerland Marco Mukrasch, Dr. Marcus
Zweckstetter, Prof. Christian Griesinger MPI,
Göttingen, Germany Grants from European Union,
ANR, CNRS, CEA, CCRT, NIH
66
Conformational Behaviour of ??????????? from RDCs
Mukrasch, Markwick, Biernat, von Bergen, Bernado,
Griesinger, Mandelkow, Zweckstetter, Blackledge 
J.Am.Chem.Soc. 129, 5235-5243 (2007).
67
Conformational Behaviour of ??????????? from RDCs
Mukrasch, Markwick, Biernat, von Bergen, Bernado,
Griesinger, Mandelkow, Zweckstetter, Blackledge 
J.Am.Chem.Soc. 129, 5235-5243 (2007).
68
Comparison of Averaged Properties of Calculated
Ensembles
Kratky plots
ltRgyrgt
Complete protein with different tail models
Poly-Gly 22Å
Extended 68Å
?/? propensity 28Å
Experimental 29Å
Model reproduces the average mass distribution of
the system
69
No contact
Predicted effect of long-range distances on RDCs
from 140 residue poly-Alanine
70
How general is this model ?
Rgyr measured in several proteins (8 to 268
residues) under conditions of high denaturant
compared to predicted Rgyr using the
flexible-meccano simulation approach
Kohn et al. PNAS 2004, 101, 12491.
71
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Comparison of calculated and experimental
couplings NHNH (i1), NHNH(i2), NH?H ,
NH?H(i-1)
Optimal scaling factors are non-uniform for
different 1H-1H couplings
72
Conformational Behaviour of Urea-Unfolded
Ubiquitin
Comparison of calculated and experimental
couplings
Database 30 extended
Database 20 extended
Increasing sampling of extended conformations
Database 10 extended
Coil Database
73
Solution NMR Timescales for Biomolecular Motion
ps
ns
?s
ms
s
Spin relaxation
Lineshape/exchange
Relaxation dispersion
Dipolar, scalar couplings
Enzyme catalysis Signal transduction Ligand
binding Collective motions?
Librational motion Normal modes Rotational
diffusion
Protein folding Hinge Bending
74
Conformational Sampling of Unstructured Domain of
Protein X
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
?
?
75
Conformational Sampling of Unstructured Domain of
Protein X
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
?
?
76
Conformational Sampling of Unstructured Domain of
Protein X
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
?
?
77
Conformational Sampling of Unstructured Domain of
Protein X
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
?
?
78
Conformational Sampling of Unstructured Domain of
Protein X
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
?
?
79
Conformational Sampling of Unstructured Domain of
Protein X
Zweckstetter Bax J.A.C.S. 2000, 122, 3791
Almond Axelsen J.A.C.S. 2002, 124, 9986.
80
Conformational Sampling of Unstructured Domain of
Protein X
Creation of a conformational ensemble Building
conformers from ?/? restraints using
flexible-meccano
?
?
81
Residual Dipolar Couplings
15N
1H
Residual dipolar couplings report on the
orientation of internuclear vectors relative to
molecular alignment tensor
82
RDCs measured in ?-Synuclein
83
Conformational Behaviour of Natively Unstructured
Proteins The Importance of Being Disordered

• Many proteins fold only on interaction with
  physiological partners - Mechanisms and
  thermodynamics of coupled folding and binding?
• Conformational plasticity enables high binding
  specificity combined with low affinity, binding
  to different partners, with increased surface
  area
• How unstructured is the protein? Importance of
  transient long-range contacts (protection
  against proteolysis, fibrillation)