Dipolar Coupling: RDCS, Structure, Dynamics and Disorder! Embo Course 2013!

EMBO NMR Course 2013 Basel
RDCs, Structure, Dynamics and Disorder!
EMBO Course 2013!
Martin Blackledge!
Protein Dynamics and Flexibility by NMR!
Institut de Biologie Structurale!
Grenoble!
Dipolar Coupling
B0 Isotropic liquid Anisotropic liquid
!
r
Dipole-dipole interaction
between two magnetic
moments
" i" j 0 h P2 (cos$ (t ))

Dij = ! 3 3 Incomplete orientational
8# rij Coupling averaged sampling - residual
to zero dipolar coupling
B0!
= 0!
Time and ensemble average!
Dipolar coupling averages to zero when all orientations ! are equally probable!
B0!
" 0!
Contains information about molecular restriction, and orientation of the dipole within molecular frame!
Non-zero average when all orientations ! are not equally probable!

It is useful to transform from the

laboratory to the molecular frame!
!"
Consider a dipolar spin pair under conditions of partial alignment in solution!
!"
Assuming the molecule is globular and rigid, we can express dipole orientation ! in terms
of molecular reorientation relative to the field and local vector orientation relative to
molecular frame!
molecular frame!
molecular frame!
of molecular reorientation relative to the field : #$ and local vector orientation relative to
molecular frame:%$!
Ci=cos#$!
ci=cos%$!
A!
We can then
Notedescribe the preferential
that averaging concernsorientational averaging
the molecular of the
orientation molecule
relative to theinfield
terms
! of a
symmetric
TwoOrder
spinsmatrix A comprising
are therefore products
assumed of thethe
rigid within averaged angular
molecular frameterms
! !!i
Order
We can then (Saupe)
describe thematrix A measures
preferential the level
orientational and direction
averaging of theofmolecule
alignment
in!terms of a
A is symmetric
a traceless Order
secondmatrix
rank tensor (Alignment tensor) and has 5 independent elements.
A comprising products of the averaged angular terms !!i !
R {&,',(}!
It is convenient to use the Principal Axis System (PAS) in which the off-diagonal elements of the
Order matrix are zero (diagonalisation). The orientation of the PAS can be defined relative to the
molecular frame using an Euler rotation R{",#,$}%
*xx+ *yy+ *zz= 0; "

*a= *zz/2 : Axial component; !
*r= (*xx - *yy) )/3 : Rhombicity/anisotropy! Azz!
Axx!
Ayy!
Dipolar couplings can then be simply expressed in terms of the vector orientation {!,)} in the
eigenframe of the tensor, and the eigenvalues of the tensor, Axx, Ayy and Azz%
Equivalent descriptions!
Azz!
Axx!
Ayy!
*a, *r, {&,',(}"
Order matrix A is a traceless second rank tensor (also known as the Alignment tensor) and has
five independent elements.!
5 alignment tensor elements can be determined from >5 independent coupling measurements!
Azz!
Axx!
Ayy!
. . . . . . .
Order matrix A is a traceless second rank tensor (also known as the Alignment tensor) and has
five independent elements.!
Structural Information from Residual Dipolar Couplings!
Azz
Residual dipolar
couplings
Alignment framereport on
(tensor)
defined
thebyorientation
5 independent
of
parameters - (Aa,Ar,",#,$)!
internuclear vectors
relative to molecular
Axx alignment tensor!
Ayy
Azz
Dmax!
Alignment frame (tensor)
Available
defined by 5 independent
parameters - (Aa,Ar,",#,$)
orientations of !
internuclear bond for
measured RDC!
Axx
Dmin!
Ayy
Steric Alignment!
Bicelles!
Alcohol mixtures!
Strained Gels!
..!
Electrostatic !
Alignment!
Bacteriophage!
Charged bicelles!
Purple membranes!
.!
Anisotropic Interactions in Solution State NMR :!

Applications to Biomolecular Structure and Dynamics!
" !Incomplete averaging of the dipolar interaction!
" !Structural information available from dipolar couplings !
" !Combination with structural databases : Fold validation/recognition!
" !Molecular domain orientation/complexes using dipolar couplings!
" !Application of dipolar couplings to de novo structure determination!
" !Structure refinement using a hybrid potential energy function !
" !Dynamic information from dipolar couplings!
" !Dipolar couplings in unstructured proteins!

Introduction of Structural Coherence!
Azz!
Axx!
Ayy!
Available orientations of interaction vector for Available orientations for chiral motif
measured RDC! of known structure!
Al-Hashimi et al J. Magn. Reson. 2000, 143, 402-406.!

Introduction of Structural Coherence!
Azz!
Axx!
Ayy!
Available orientations of interaction vector for Available orientations for chiral motif
measured RDC! of known structure!
Al-Hashimi et al J. Magn. Reson. 2000, 143, 402-406.!
Residual Dipolar Couplings !
15N!
II!
III!
I!
II!
I!
III!
Resonance Frequency ! 1H!

Rapid Fold Identification : Database Long-range structure determination in

comparison! extended molecules!
Ab initio structure determination!
RDCs for Structure !


Long-range Conformational
restraints from RDCs!
Orientation of structural domains

relative to a common reference frame!
Example - !
" Measurement of RDC in modules

of known structure!
" Determine alignment axes!
" Superpose axes!
Orientation of structural domains using Dipolar Couplings!
Structural changes associated with domain reorientation in MBP
E.g.: Millet, et al. (2003) Proc. Natl. Acad. Sci. USA 100, 12700-12705
Orientation of Interaction Partners in Molecular Complexes!
In combination with chemical shift mapping,

intermolecular nOe, paramagnetic effects!
E.g. : Clore & Schwieters (2003) J. Am. Chem. Soc. 125, 2902
Orientation of structural domains using Dipolar Couplings!
The Global Conformation of the Hammerhead Ribozyme Determined Using

Residual Dipolar Couplings!
Bondensgard et al. (2002) Biochemistry 41, 11532

Tools for analysing and exploiting Dipolar Couplings!
" !SVD ! !Losonczi et al, J. Magn. Reson., 138 (1999) 334!
" !Dipocoup !Meiler et al, J. Biomol. NMR, 17 (2000) 283.!
" !PALES !Zweckstetter, Bax, J. Am. Chem. Soc., 122 (2000) 3791.!
" !REDCAT !Valafar, Prestegard, J. Magn. Reson., 167 (2004) 228.!
" !MODULE !Dosset et al, J. Biomol. NMR, 20 (2001) 223.
" !.!
All search {Aa, Ar, &, ', (} parameters, minimizing against target function :
Allow determination of alignment properties, rotation of coordinates into alignment frame and
comparison of quality of data reproduction from structural models
MODULE : A graphical tool for analysing and exploiting RDC!
Alignment
tensor
determination
and rigid body
molecular!
modeling!
Dosset et al (2001) J
Biomol NMR, 20, 223.!
MODULE is available
at http://www.ibs.fr!
Searches {Aa, Ar, ", #, $}parameters for

each tensor, minimizing target function :!
Aligns modules by orienting !

Eigenframes into a common !
reference frame!
MODULE : A graphical tool for

analysing and exploiting RDCs!
MODULE : A graphical tool for analysing and exploiting RDC!
&x! &y! &z!
Aligns modules by orienting ! Presents 4 degenerate orientational solutions!

Eigenframes into a common !
reference frame.!
Effect of differential
domain mobility !
Differential mobility
gives rise to different
alignment
characteristics in
different parts of the
molecule!
This can give

important information
about!
domain motions!
E.g. Tolman et al Nat. Struct. Biol. 1997, 4, 292. Fischer et al 1999, Biochemistry 38, 9013-9022, Skrynnikov et al J.MB 2000, 295, 1265
Tolman et al, J. Am. Chem .Soc. 2001, 123, 1416-1424. Lukin et al, PNAS, 2003, 100, 26174
Structures of protein complexes in the weak binding regime!
Dijexp = pbound Dijbound + (1" pbound )Dijfree
Bolon et al (1999) J. Mol. Biol. 293 107-115, Koenig et al (2002) J. Mol. Biol. 322 441-461!
!
Titration of RDCs for accurate determination of protein complexes!
{
Dijm,exp = "m Dijm = "m pbound Dijbound + (1# pbound )Dijfree }
! CD2AP SH3!
Ubiquitin!
pbound!
pbound! pbound!
Titration of RDCs for accurate determination of protein complexes!
{
Dijm,exp = "m Dijm = "m pbound Dijbound + (1# pbound )Dijfree }
CD2AP SH3!
!
1D 1D
HN CaC
Ubiquitin!
1D
HN
1D 2D
CaH HNC
a
Cross validation of 10% RDCs not used in 'm optimization!

The importance of being saturated!
Rotation relative to
pbound = 1.00!
correct orientation
pbound = 0.90! ("=10, (# =10, ($ =2%
pbound = 0.80! ("=32, (# =20, ($ =7%
pbound = 0.70! ("=54, (# =29, ($ =12%


Steric Alignment!
Bicelles!
Alcohol mixtures!
Strained Gels!
..!
Electrostatic !
Alignment!
Bacteriophage!
Charged bicelles!
Purple membranes!
.!
Structural information available from dipolar couplings!
Raising orientational degeneracy

using different alignment media!
Continuum degeneracy for single

vector raised to 8 equivalent
orientations in presence of two
tensors!
Ramirez & Bax, JACS 1998 120 , 9106!

Structural information available from dipolar couplings!
Raising orientational degeneracy using different alignment media!
structure! !vector! chiral!
1 tensor!
!#! 4!
(1 medium)!
2 tensors!
!8! 1!
(2 media)!
Protein backbone comprised of connected $planar% elements!
Orientational definition of geometric objects from RDCs!

from two alignment media!
structure! vector! plane! chiral!
2 tensors! 8! 2! 1!
Construction of backbone fold!

from oriented peptide units!
H N! C"!
H N!
H N!
C"!
C"!
C"!
Meccano!
NHN!
C%HN!
C%N!
C%C"!
C#C"%
H"C"%
Sequence!
Experimental coupling!
Determination of protein backbone structure using only

residual dipolar couplings - Meccano!
Medium 1 ! !Medium 2!
63 N-HN ! !63 N-HN!
61 C%-HN ! !61 C%-HN!
61 C%-N ! !63 C%-N!
59 C"-C% ! !54 C"-C%!
62 C"-H" ! !62 C"-H"%
39 C"!C# % % %%
% % % %Cornilescu et al J.A.C.S. 1998, 6836; Ottiger & Bax JACS 1998, 12334!
Alignment Medium 1! Alignment Medium 2!
Pro196!
Systematic
FOCUSSED
Cys200! violation of RDC in
STRUCTURE
residues 196-203
DETERMINATION
Gly203! USING MECCANO
DNH!
+25!
Determination of the active site
conformation of MsrA using only RDC!
-25!
+8!
DC%HN!
-8!
DC%C"!
+5!
203!
-5!
196!
DC%C#!
+5!
Phage!
Application of
C12E6/hexanol!
MECCANO to define -5!
196 197 198 199 200 201 202 203!
local structure! Sequence!

Determination of the
active site
conformation of
MsrA using only
RDC!
Visualisation using
MODULE!
Meccano fragment
and MsrA
conformation share
common alignment
frames! Phage!
active site
conformation of
MsrA using only
RDC!
Visualisation using
MODULE!
Meccano fragment
and MsrA
conformation share
common alignment
frames! Phage Medium!
active site
conformation of
MsrA using only
RDC!
Visualisation using
MODULE!
Meccano fragment
and MsrA
conformation share
common alignment
frames! Phage Medium!
Determination of the active site conformation of MsrA!
RDC-defined structure!
Cys200!
Cys53!
Msr (E.coli)!
Application of MECCANO to define local structure !


Residual dipolar couplings as constraints for structure refinement!
" Include RDC target function into

a physical force field!
" Drive molecule into a

conformation in agreement with
orientational restraints1.!
" Angular dependence requires

strong local geometric restraints
(planarity, covalent angles).!
1. Clore & Gronenborn, 1998, PNAS, Garrett et al, 1999, Tsui et al 1999, Hus et al 2000.!
2. Meiler et al 2000, J.Biomol.NMR , 245-252. !
3. Moltke & Grzesiek, 1999, J.Biomol.NMR 15, 77-82.!
Apart from improving coordinate rmsd, how much information can

we derive from a single set of RDCs?!
16 different planar orientations are in agreement with RDCs!

from a single alignment medium!
Hus et al J.A.C.S. (2008).
structure! vector! plane! chiral!
1 tensor!
#! 16! 4!
(1 medium)!
2 tensors!
8! 2! 1!
(2 media)!

Refinement using RDC data sets simulated from 2

alignment media in combination with full experimental
nOe data set!
Comparison with a third $free% data set from an

independent alignment tensor!
Correlation with $free% alignment medium data set!

No RDCs! 1 medium! 2 media! 2 media!
(nOe only)! N-HN + nOe! 3 pp RDCs! 3 pp RDCs! NH only!
Correlation with $free% alignment medium data set!

1D + nOe!
N-HN
Top ten energetically equivalent
(RDC/nOe/total) structures determined using
single set of NH RDCs in combination with
full set of experimental nOes

Simulated Dij (Hz)
Experimental Dij (Hz)

Complete set of peptide plane RDCs!
Equivalent orientations of peptide planes in

agreement with single alignment tensor data
sets!
(red : correct orientation, blue : incorrect
orientation)!

Experimental case!
Refinement using full peptide plane
experimental data from single alignment media!
Equivalent orientations of peptide planes in

agreement with single alignment tensor data
sets!
(orange tensor A!
blue tensor B)!
Hus et al J.A.C.S. (2008).


Characteristic NMR timescales
ps ns s ms s
Spin relaxation Lineshape/exchange
Relaxation dispersion
Real-time NMR
Dipolar, scalar couplings, chemical shifts
Librational motion Enzyme catalysis Protein folding

Normal modes Signal transduction Hinge Bending
Rotational diffusion Ligand binding
Collective motions?
Simultaneous determination of protein structure and dynamics from NMR!
Motional properties are often measured

independently of molecular conformation!
Most structures are determined assuming single

conformers to describe dynamic average!
Simultaneous determination of both structure and

dynamics would provide essential details of the
conformational behaviour of the protein!

Most structures are determined assuming single


Ensemble averaged restrained molecular dynamics:

Mutli-copy description describes the data better than a single structural model
Dynamic averaging of cross relaxation (nOe) depends on nature and timescale of the motion:
ps-ns dynamics average effective distances differently to s-dynamics
Combining constraints with different time dependences mixes effective timescales
Torda, Scheek, van Gunsteren, J. Mol. Biol. 1990,214, 223. Bonvin, Rullman, Lamerichs, Boelens, Kaptein, Proteins 1993, 15, 385.!
Clore, Schwieters, Biochemistry 2004, 43, 10678. Lindorff-Larsen, Best, DePristo, Dobson, Vendruscolo, M. Nature 2005, 433, 128.!
Motional averaging to the millisecond dictates

interpretation of all NMR data - Chemical shift!
Chemical shift alone cannot define the nature of the

conformational average (yet?)!
Residual dipolar couplings average on the same

timescale as chemical shift: may hold the key to
resolving the conformational sampling problem!
15N
ps ns s ms s
Spin relaxation
Real time
Librational motion Enzyme catalysis Assembly

Rotational diffusion Signal transduction
Ligand binding 1H
Ci=cos#$!
ci=cos%$!
Note that averaging concerns the molecular orientation relative to the field!
Two spins are therefore assumed rigid within the molecular frame!
Azz!
Axx!
Ayy!
If we assume
In thethe motion of
presence exerts negligible
motion influence
coupling on the
is averaged molecular
over time andalignment
ensemble tensor
:! A,
averaging can be restricted to terms concerning orientation of the internuclear vector!
Where!
$ 3cos 2 ! # 1 3 '
D jk (! ," ) = bij & Aa + Ar sin 2 ! cos 2" )
and pn is the population&% 2 4
of conformation n in the average
)( !
Dynamic averaging of dipolar couplings in weakly aligned systems
Report on average of
all!
conformations
sampled up to ms
timescale!
-!
Characterisation of
slower motions in
proteins!
Report on average of
all!
conformations
sampled up to ms
timescale!
-!
Characterisation of
slower motions in
proteins!
Report on average
of averaging
Different all!
effects found with
conformations
different alignment
sampled up to ms
media !
timescale
-
-!
Elucidation of local
Characterisation of
motional modes and
slower motions in
amplitudes
proteins!
$ 3cos 2 ! # 1 3 '
D jk (! ," ) = bij & Aa + Ar sin 2 ! cos 2" )
&% 2 4 )(
Meiler et al J.A.C.S. 123, 6098 (2001). Briggman & Tolman J.A.C.S. 125, 10164 (2003), !
Ulmer et al J.A.C.S. 125, 9179 (2003), Bernado & Blackledge J.A.C.S. 126, 4907 (2004) !
(",#,$)%
> 5 different media : Determination of all 5 averaged spherical harmonic

terms
Meiler et al J.A.C.S. 123, 6098 (2001).
Anisotropic motion
" i " j 0 h & 3 )

3 3 ( Aa (3cos $ ! 1) +
2
Dij = !S A r sin 2$cos2%+ (1
16# rij ' 2 *
Axially symmetric motion
Collective motions from NH RDCs measured in a paramagnetically aligned protein Tolman et al Nat.
Struct. Biol. 1997, 4, 292. !
Collective motions from differential alignment behaviour of structured domains Fischer et al,
Biochemistry, 1999, 39"8, 9013, S krynnikov, Goto, Yang, Choy, Tolman, Mueller, Kay J.MB 2000, 295, 1265. !
Backbone motions from multiple RDCs in a single alignment medium Tolman et al, J.A.C.S. 2001, 123, 1416 !
Model-free backbone motion from NH RDCs in multiple (&5) alignment media Meiler, et al, J.A.C.S.
2001, 123, 6098. Peti et al J.A.C.S. 2002, 124, 5822. Tolman, J.A.C.S. 2001, 124, 12020. !
Backbone structure and dynamics from multiple RDCs in multiple alignment media!
Ulmer, et al, J.A.C.S. 2003, !
.!

Most structures determined assuming single


RDCs are well suited to

simultaneous determination
of structure and dynamics
providing direct, analytical
determination of both
components!
Approaches to Determination of Protein Dynamics from RDCs!

Direct analysis of RDCs:! Molecular dynamics simulation
Fitting of motional modes and Comparison with MD, RDC as
amplitudes to experimental data! restraints to determine compatible
conformations/ensembles!
Advantages : Modelfree! Advantages : Molecular description!

Disadvantages : Abstract description Disadvantages : Effects of combining
Stability/robustness, effects of noise, force field and constraint terms, non-
absolute calibration! Boltzmann statistics!
Tolman, Griesinger, Brschweiler, Bax, Al Hashimi Clore, Vendruscolo, Grubmller, Brschweiler, Salvatella, Al Hashimi


Stability /robustness, effects of noise, force field and constraint terms, non-
3DGAF - A Local Motional Model for Backbone Dynamics
NH!
"C !
i
N! C! "C !
i-1
O!
Diffusive motions about three

perpendicular axes (3D-GAF)
Bremi & Brschweiler J.A.C.S. 119, 6672 (1997)!

Bouvignies, Bernado, Meier, Cho, Grzesiek, Brschweiler & Blackledge P.N.A.S. 102, 13885 (2005)
General Model of Local Backbone Motion in Proteins
Up to 6 different RDCs measured in 7 alignment systems in

protein GB3 (27 RDCs/peptide)
Backbone motions in an Immunoglobin binding domain of Streptococcal

protein G
Ulmer et al J.A.C.S. 125, 10164 (2003), Meier et al J.A.C.S. 125, 44-45 (2003)
Modelling of 3D GAF amplitudes in protein GB3
Crystal
structure
"C !
Motionally
"C !
i i-1
averaged RDCs
Anisotropic motional model

Quantitative Determination of ps-ms Backbone Motion in Ubiquitin from RDCs
Data Fitting Protocol
Target function :
!,),+ mean orientation of plane, A alignment tensor, ,&, ,' , ,( , S motional amplitudes
Explicit GAF averaged dipolar interaction

R(!,),+)
"C ! "C !
i i-1
3D Gaussian Axial Fluctuation Model
Quantitative Determination of ps-ms Backbone Motion in Ubiquitin from RDCs
Molecular Alignment tensor : Local Motion :

Reference frame for dynamic averaging Dynamic amplitudes and directions
Dynamically averaged RDC!
Estimation of absolute level of molecular alignment essential for quantitative dynamic analysis


Stability /robustness, effects of noise, force field and constraint terms, non-


Conformational Behaviour of Intrinsically Unfolded Proteins

The Dark Side of Structural Biology!
Intrinsically disordered regions present in 30-40% of proteins coded in eukaryotic genomes!
Implicated in transcriptional regulation, !

translation and cellular signal transduction, !
post-translational modification,
molecular recognition,
cell cycle control, !
cancer-related, as well as
neurodegenerative pathology!
When a protein is not folded in its biologically active form:!

How do we understand its molecular function.?!
Dyson & Wright Nature Rev Mol Cell Biol, 6, 197, 2005; Tompa FEBS Lett 579, 3346,2005, Dunker et al Biochemistry 5674, 41, 2002,
Conformational Behaviour of Intrinsically Unstructured Proteins !

A Challenge for Structural Biology!
How does primary sequence code for function
in a highly flexible protein?
Intrinsically disordered proteins undergo

(ppm)
exchange between many different

conformations in solution:
15N
Ensemble description is appropriate
1H (ppm)
NMR provides site specific,
ensemble-averaged structural
parameters
Secondary chemical shift : Paramagnetic Relaxation Enhancement :

((C"% Probe for local structural propensity A Probe for Transient Long-range Structure
helix
1.0 no contacts
I/I0
0
((C#% SSP Sequence
2
sheet 1 1 $ 0 ' 2 2 2
"2 = & ) * I g B s( s +1){4J(0) + 3J(+ I )}
r 6 15 % 4 # (
!
Marsh et al Protein Sci. 2006; 15: 27952804
Characterisation of unfolded! Residual dipolar couplings depend on

angular averaging properties
proteins using NMR of bond vectors
ps ns s ms s )%
Spin relaxation
Real time
RDCs in Unfolded Proteins! B0
)%
Averaged dipolar couplings values

depend on averaging of bond vector
orientation relative to B0 field
PPII
"
Helical regions are characterised by positive RDCs
Under denaturing conditions Partially unfolded (pH 2.3)

RDCs in Unfolded Proteins! B0
)%
Averaged dipolar couplings values

depend on averaging of bond vector
orientation relative to B0 field
Effect of local structure on RDC profiles
Averaged dipolar couplings sensitive to sampling of bond vector

orientations relative to B0 field
An ensemble description of the unfolded state: Flexible Meccano !
K R D E P .
+%
*% .
.!
<Dij>
Calculation of average RDCs over

conformational ensemble!
Amino acid specific sampling from a coil database combined with steric exclusion!
Bernado, Blanchard, Timmins, Marion, Ruigrok & Blackledge Proc.Natl.Acad.Sci. 102, 17002 (2005)
Conformational Sampling of Unstructured Proteins!

.!
Calculation of average Dipolar

Couplings for conformational
<Dij>
ensemble!
Conformational Sampling of Unstructured Domain of Protein X
.!
Calculation of average dipolar couplings for conformational ensemble
Protein X from Sendai virus - containing

both folded and unfolded domains
<DN-NH>
+%
Random */+ sampling

Simple non-bonding exclusion
*% Bernado, Blanchard, Timmins, Marion, Ruigrok & Blackledge Proc.Natl.Acad.Sci. 102, 17002 (2005)

Sampling */+ using amino acid

specific propensities
Simple non-bonding exclusion
Ala Pro Leu ! Bernado, Blanchard, Timmins, Marion, Ruigrok & Blackledge Proc.Natl.Acad.Sci. 102, 17002 (2005)

Model quantitatively reproduces distribution and proportion of

dipolar couplings in folded and unfolded domains
RDCs: Probes for local structural propensity
.
C"C% NH-N C"H"%
i+1
*%
. +%
C"C% NH-N C"H"% i-1
.
C"C% NH-N C"H"%
Predicted dependence of RDCs on local and immediate neighbours
Random Coil RDC Values from Unfolded Proteins !
C terminal partially folded domain!

of Sendai virus Phosphoprotein!
Bernado et al Proc.Natl.Acad.Sci. 102, 17002 (2005)
Staphyloccocal Nuclease ,131, in Urea!

Shortle & Ackerman Science 2001, 293, 487.
Urea-denatured apo-myoglobin!
Mohana-Borges et al JMB 2004, 340, 1131.!

Random Coil RDC Values from Unfolded Proteins !
A model for expected random

coil dipolar couplings from
unfolded chains based on
primary sequence!
Ensemble Descriptions of Highly Disordered Polymers!
" !Biophysical Data ! Ensemble Description!
" !Large number of degrees of freedom: Limited experimental data!
" !Is the solution unique?!
" !Classical ill-posed problem!
" !Refinement against experimental data may not deliver statistical

!mechanical ensembles!
" !Agreement with experimental data may not guarantee physically

!meaningful ensembles !
Jacques Hadamard (1902): Sur les problmes aux drives partielles et leur
signification physique. Princeton University Bulletin, 49--52.
RDCs
1D
Ensemble Description of! CaHa
1D
HN
Disordered Proteins
-D
HNHN
DHNHa
15N/1H PREs
Ensemble selection to match experimental

data within estimated uncertainty
Nodet et al, J. Am. Chem. Soc. 131, 17908 (2009),

Jensen et al J. Am. Chem. Soc, 132, 1270, (2010),
Salmon et al, J. Am. Chem. Soc, 132, 8407 (2010)
Chemical shifts
How well can we define the potential energy landscape of IDPs using NMR?
*% Predicted CSs and RDCs for Alanine tri-

+%
peptide in statistical coil conformations
EMBO NMR Course 2013 Basel -P (compared to statistical coil) red +ve, blue -ve
Target
13C&, 13C', 13C
13C&, 13C', 13C"

15N , 1H
1D 2D
NH, CNH, !
1D , 1D
CaHa CaC#
13C&, 13C', 13C"
+ 1DNH
13C&, 13C', 13C, !

NH,
1D 2D
CNH, !
1D 1D
CaHa, CaC#
Mapping the potential energy landscapes!

of IDPs at amino acid specific level
Ozenne V, et al J Am Chem Soc. 2012 134:15138-48
Flexible-Meccano
Available with user-friendly interface at:
http://www.ibs.fr/science-213/scientific-output/software/flexible-meccano
Features include:
" Efficient ensemble generation of IDPs (PDB files)
" Ensemble calculation of RDCs, PREs and J-

couplings from user-defined sequence
" Possibility for testing specific conformational

sampling regimes
" Scripts provided for interfacing to SPARTA

(chemical shift prediction) and CRYSOL (small
angle scattering curves)
Ozenne, Bauer, Salmon, Huang, Jensen, Segard, Bernado, Charavay, Blackledge (2012) Bioinformatics 28, 1463-1470
Characterisation of the conformational behaviour of IDPs using RDCs
Agadir
RDCs can tell us more about the helical properties of the partially folded chain
Identification of helical conformations present in Ntail molecular recognition

element in solution
RDCs predicted from 153

statistical coil ensembles
Each including helical
segments from 4-20 amino
acids in length situated
between amino acids 473
and 496
...
...
...
Characterisation of the conformational behaviour of the pre-recognition

state of Ntail using NMR residual dipolar couplings
Dij,exp = Dij,unfolded
...
...
Combination of the different ensemble

patterns to best reproduce experimental
data

-2=./((Dij,eff -Dij,exp ) /0ij) 2% -2=427!
Dij,eff = Dij,unfolded
Dij,eff = p1Dij,hel1+ (1-p1)Dij,unfolded
Dij,eff = p1Dij,hel1+ p2Dij,hel2+ (1-p1- p2)Dij,unfolded
Dij,eff = p1Dij,hel1+ p2Dij,hel2 + p3Dij,hel3 + (1-p1-p2-p3)Dij,unfolded
...
...

data

-2=./((Dij,eff -Dij,exp ) /0ij) 2% -2=227!
Dij,eff = p1Dij,hel1+ p2Dij,hel2+ (1-p1- p2)Dij,unfolded
...
...

data

-2=./((Dij,eff -Dij,exp ) /0ij) 2% -2=120!
Dij,eff = p1Dij,hel1+ p2Dij,hel2+ (1-p1-p2)Dij,unfolded
No populations -2/N conformers significance

...
...
1 427/96 476-488
2 227/93 476-488,479-484 p<0.0001
476-488,479-484,
3 120/90 p<0.0001
478-492
476-488,479-484,
4 113/87 p=0.16
478-492, 479-492

state of NTAIL using NMR residual dipolar couplings
Minimum
...
Ensemble
Description

Sendai virus
Molecular recognition element samples specific helical conformers
Unfolded H1 (479-484)
(255)% (364)%
Disordered domain
of Nucleoprotein: NTAIL
H3 (478-492) H2 (476-488)
(119)% (286)%
..EEETNDEDVSDIERRIAMRLAERRQEDSATHGDE.. !
..EEETNDEDVSDIERRIAMRLAERRQEDSATHGDE.. ! Fraying is not random but nucleated by helix-capping
Preferential helical sampling encoded in sequence
Jensen & Blackledge J.Am.Chem.Soc. 2008

Jensen, Lescop, Houben, Blanchard, Ruigrok, Blackledge J.Am.Chem.Soc. 2008
Jensen et al Proc Natl Acad Sci 2011

Dipolar Coupling: RDCS, Structure, Dynamics and Disorder! Embo Course 2013!

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Dipolar Coupling: RDCS, Structure, Dynamics and Disorder! Embo Course 2013!

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EMBO NMR Course 2013 Basel

RDCs, Structure, Dynamics and Disorder!

EMBO Course 2013!

Institut de Biologie Structurale!

EMBO NMR Course 2013 Basel

" i" j 0 h P2 (cos$ (t ))

Time and ensemble average!

EMBO NMR Course 2013 Basel

Non-zero average when all orientations ! are not equally probable!

It is useful to transform from the

Consider a dipolar spin pair under conditions of partial alignment in solution!

EMBO NMR Course 2013 Basel

EMBO NMR Course 2013 Basel

EMBO NMR Course 2013 Basel

EMBO NMR Course 2013 Basel

*xx+ *yy+ *zz= 0; "

EMBO NMR Course 2013 Basel

*a, *r, {&,',(}"

EMBO NMR Course 2013 Basel

Structural Information from Residual Dipolar Couplings!

Axx alignment tensor!

Structural Information from Residual Dipolar Couplings!

EMBO NMR Course 2013 Basel

Anisotropic Interactions in Solution State NMR :!

" !Incomplete averaging of the dipolar interaction!

" !Structural information available from dipolar couplings !

" !Combination with structural databases : Fold validation/recognition!

" !Molecular domain orientation/complexes using dipolar couplings!

" !Application of dipolar couplings to de novo structure determination!

" !Structure refinement using a hybrid potential energy function !

" !Dynamic information from dipolar couplings!

" !Dipolar couplings in unstructured proteins!

EMBO NMR Course 2013 Basel

Structural Information from Residual Dipolar Couplings!

Structural Information from Residual Dipolar Couplings!

EMBO NMR Course 2013 Basel

Residual Dipolar Couplings !

Resonance Frequency ! 1H!

Rapid Fold Identification : Database Long-range structure determination in

Ab initio structure determination!

RDCs for Structure !

EMBO NMR Course 2013 Basel

Anisotropic Interactions in Solution State NMR :!

" !Incomplete averaging of the dipolar interaction!

" !Structural information available from dipolar couplings !

" !Combination with structural databases : Fold validation/recognition!

" !Molecular domain orientation/complexes using dipolar couplings!

" !Application of dipolar couplings to de novo structure determination!

" !Structure refinement using a hybrid potential energy function !

" !Dynamic information from dipolar couplings!

" !Dipolar couplings in unstructured proteins!

Orientation of structural domains

" Measurement of RDC in modules

EMBO NMR Course 2013 Basel

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Structural changes associated with domain reorientation in MBP

Orientation of Interaction Partners in Molecular Complexes!

In combination with chemical shift mapping,

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The Global Conformation of the Hammerhead Ribozyme Determined Using

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xx+ yy+ *zz= 0; "

a, r, {&,',(}"