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Solution structures by NMR

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Solution structures by NMR TOCSY Total Correlation Spectroscopy Esperimento 2D analogo al COSY, utile per misurare gli accoppiamenti scalari consecutivi HNCO ... – PowerPoint PPT presentation

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Title: Solution structures by NMR


1
Solution structures by NMR
2
Structural Biology
Structure Mobility

Protein-protein Protein-ligand
interactions
NMR is a powerful method to address these problems
year
NMR structures per year
1984 1 (first structure!) 1990 25
1994 80 (first paramagnetic structure!)
1998 125 2000 200
3
Structure determination through NMR
Resonance assignment
NMR
NOE intensities and J couplings (plus other
structural constraints)
Conversion of NMR data in distances and angles
Structural constraints
Structure calculations 3D structure
Structure refinement REM, RMD
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Assignment of homonuclear spectra
8
TOCSY
  • Total Correlation Spectroscopy
  • Esperimento 2D analogo al COSY, utile per
    misurare gli accoppiamenti scalari consecutivi

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NMR Tecniques and molecular weight
14
Multifrequency NMR experiments
To make full use of multidimensional NMR, isotope
labeled samples are needed
Multifrequency NMR experiments
For each frequency dimension a different type of
coupling can be detected
15
Triple Resonance
J couplings
Relaxation rates
16
HNCO
H N - CO
Trasferisco da Hn ad N Osservo N prima
dimensione Trasferisco da N a CO Osservo CO
Seconda dimensione Trasferisco da CO a N, da N
ad Hn Osservo Hn Terza dimensione
17
Sequential asignment using the HNCA and HNCOCA
experiments
18
Assignment procedure
19
Sequential assignment in triple resonance
experiments. HNCA
13Ca
1H
Backbone assignment of 6 residues using 13Ca
20
Transfer without acquisition
21
Side chain Assignment
22
13C Chemical shift as a tool for Assignment
23
NMR structural characterization of the target
protein
  • Approaches to the Structure Determination of
    Proteins
  • For proteins of up to 30 kDa, use
    13C/15N-labelling
  • For proteins of higher molecular weight, use
    fractional deuteration and 13C/15N-labelling
  • For proteins of 100 kDa and above, use selective
    protonation and 13C/15N-labelling

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Classical constraints for structure
determination
NOE H-bonds

Distances

3J couplings Chemical shifts
?,?,?
Vector orientation

Residual dipolar couplings Cross Correlation
effects
27
Protein structure and dihedral angles
28
Contraints for Structure Calculation So far, the
emphasis has been on identification of the
observed signals in the spectra and their
correlation with the amino acid protons giving
rise to the signals. Afterwards, one has to
extract the data which are relevant for the
structure. Of special importance in this respect
are proton-proton distances, which can be
estimated from the signal intensities in the 2D
NOESY, 3D 15N-NOESY-HSQC and 3D 13C-NOESY-HSQC
spectra . Signal intensity depends on the
distance r between two nuclei i and j, according
to NOEij 1/rij6 Distances are derived from
the spectra after calibration against NOE signals
for known distances (such as distances in
elements of secondary structure) and grouped into
a few classes. An upper and a lower bound of
distance is assigned to each class. The lower
bound is often set to the sum of the van der
Waals radii of the two protons.
In this procedure, all non-sequential signals
which are visible in the NOESY spectra have to be
assigned, the number of which easily exceeds 1000
in a medium-sized protein (ca. 120 amino acids).
It is distinguished between cross peaks of
protons no more than five amino acids apart in
the protein sequence (medium range NOE's) and
those which are more than five amino acids apart
(long range NOE's). The former are mainly
indicative of the protein backbone conformation
and are used for secondary structure
determination, whereas the latter are an
expression of the global structure of the protein
and therefore contain the main information used
for tertiary structure calculation. In addition
to interproton distances the phi-dihedral angles
of the protein backbone can be determined from a
COSY spectrum or a HNCA-J spectrum (a variant of
the HNCA spectrum, from which the coupling
constants of the N-Calpha bonds can be
determined). Dihedral angles are connected with
the coupling constants via the Karplus equation .
29
Protein structure and dihedral angles
30
Calculation of 3D protein and nucleic acid
structures
The program DYANA
Simulated annealing combined with molecular
dynamics in torsion angle space

Numerical solution of the classical mechanical
Lagrange equations of motion with torsion angles
as generalized internal coordinates
The NMR constraints are used as pseudopotential
to calculate the velocity
A temperature bath to cross barriers between
local minima is cooled down slowly from its
initial high temperature
The target function represents the potential
energy of the system
other constraint contributions
Güntert P., Mumenthaler C., Wüthrich K., J.Mol.
Biol., 1997
31
Structure quality through PROCHECK
  • Covalent geometry
  • Torsion angles
  • Chirality
  • Planarity
  • Precision
  • Restraint violations

Results are presented as plots suitable for
publication
Laskowski R A, MacArthur M W, Moss D S Thornton
J M (1993). J. Appl. Cryst., 26, 283-291.
32
Classical constraints for structure
determination
NOE H-bonds

Distances

3J couplings Chemical shifts
?,?,?
Vector orientation

Residual dipolar couplings Cross Correlation
effects
33
Strategies for Sequential Assignment
Using this cyclic procedure of alternatively
connecting intraresidual TOCSY with
interresidual NOESY cross peaks one can walk -
ideally - along the entire length of the
protein. Problem there are a few proline
residues in most proteins. Problem there are a
number of additional short proton proton
distances which can occur as a result of certain
elements of secondary structure.
The general work of Wuthrich and co-workers
identified a whole range of secondary specific
short proton proton distances that are
summarized here
34
Strategies for Sequential Assignment
Here are a number of characteristic distances
that connect the two strands of a b-sheet short
enough to appear as cross peaks in a NOESY
spectrum.
These are a- a, amide- a and amide-amide
distances
  • b-sheet specific NOEs in red and simple
    sequential NOEs in green.
  • Other regular elements of secondary structure,
    e.g. different types of
  • -turns, 3-10 helices and parallel b-strands, are
    characterized by similar
  • patterns of short distances involving backbone
    protons.

35
Calculation of Tertiary Structure
Results - The Structure Family After the
structural calculations a family of structures is
obtained instead of an exactly defined structure.
This family spans out a relatively narrow
conformational space. Therefore, the quality of a
NMR structure can be defined by the mean
deviation of each structure from this family
(RMSD) from an energy minimized mean structure
which has to be calculated previously. The
smaller the deviation from this mean structure
the narrower the conformational space. Another
definition of RMSD is to compare pairwise the
structures of a family and calculate the mean of
these deviations. The RMSD is different for
different parts of the protein structure Regions
with flexible structure or without secondary
structure (loops) show a larger deviation than
those with rigid and well defined secondary
structure. This higher RMSD in loops results in
first instance from the smaller number of
distance constraints for these parts of the
protein structure. Additionally it can originate
from real flexibility, but this diagnosis can
only be confirmed by measuring the relaxation
times for the protein. A result of a structure
calculation is shown here
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Calculation of Tertiary Structure
37
Calculation of Tertiary Structure
The idea of computer-aided structure calculation
is to convert distance- and torsion-angle-data
(constraints) into a visible structure. However,
the experimentally determined distances and
torsion angles by themselves are not sufficient
to fully characterize a protein structure, as
they are based on a limited number of
proton-proton distances. Only the knowledge of
empirical input data, such as bond lengths of all
covalently attached atoms and bond angles,
enables a reasonably exact structure
determination.
38
Calculation of Tertiary Structure
For this purpose, a randomly folded starting
structure is calculated from the empirical data
and the known amino acid sequence. The computer
program then tries to fold the starting structure
in such a way, that the experimentally determined
interproton distances are satisfied by the
calculated structures. In order to achieve this,
each known parameter is assigned an energy
potential, which will give minimal energy if the
calculated distance or angle coincides with its
input value. The computer program tries to
calculate a structure with a possibly small
overall energy.
39
Calculation of Tertiary Structure
Without the experimentally determined distance-
and torsion angle-constraints from the NMR
spectra, the protein molecule can adopt a huge
number of conformations due to the free rotation
around its chemical bonds (except for the peptide
bond, of course). the N-Calpha bond and the
Calpha-CO bond. All these possible conformations
are summed up in the so-called conformational
space. Therefore, it is important to identify as
many constraints as possible from the NMR spectra
to restrict the conformational space as much as
possible, thus getting close to the true
structure of the protein. In fact, the number of
constraints employed is more important than the
accuracy of proton-proton distances, so that the
classification above is sufficiently precise.
40
Calculation of Tertiary Structure
Energy Potentials A starting structure is needed
for a molecular dynamics calculation, which is
generated from all constraints for the molecular
structure, such as bond-lengths and bond-angles.
This starting structure may be any conformation
such as an extended strand or an already folded
protein. During the simulation, it develops in a
potential field under the influence of various
forces, in which all information about the
protein is summarized. Two classes of energy
terms are distinguished Eempirical and
Eeffective V Eempirical Eeffective with
Eeffective ENOE Etorsion, and Eempirical
Ebond Eangle Edihedral Evdw Eelectr
Eempirical contains all information about the
primary structure of the protein and also data
about topology and bonds in proteins in general.
                                  The
contributions of covalent bonds, bond-angles and
dihedral angles towards Eempirical are
approximated by a harmonic function. In contrast,
non-covalent van-der-Waals forces and
electrostatic interactions are simulated by an
inharmonic Lennard-Jones potential or Coulomb
potential, respectively. Eeffective takes the
experimentally determined constraints into
account. Angle constraints are introduced by a
harmonic function analogous to that for the
dihedral angles. For distance constraints, the
energy potential will be set to zero, if the
corresponding distance is within the given
limits. If it is outside these limits, a harmonic
energy potential is used, which tries to push the
value of the distance into the limits.
41
AGGFHRLIFTHWQDCSAAVHYLGGP..
Ogni aminoacido ha valori precisi di Distanze
tra atomi.
Libreria
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Sequenza primaria Libreria di aminoacidi Legame
peptidico 180 Distanze tra protoni
intraresiduo Distanze tra protoni di residui
consecutivi Distanze tra protoni di residui a
breve distanza (i,i4) Angoli diedri y
j Distanze tra prootni a madio e lungo raggio
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Cosa Ottengo?
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Target (penalty) Function
Ripeto il calcolo n volte Per Ogni struttura
calcolo il valore della funzione
penalità Seleziono le strutture che hanno il
piu basso valore della funzione penalità
50
Target (penalty) Function
La somma delle violazioni dei vincoli sperimentali
E di fatto, impossibile ottenere una struttura
che sia in grado di rispettare perfettamente
linsieme di tutti i vincoli sperimentali che noi
imponiamo
Non ci sono solo I vincoli sperimentali, ma
quelli derivanti dalla struttura di un
polipeptide, (es le violazioni di Van der Walls,
gli angoli non permessi, etc..)
51
Target (penalty) Function
La somma delle violazioni dei vincoli sperimentali
Considero buone tutte quelle strutture che
hanno il più basso valore della funzione penalità
52
Famiglia di strutture
Perché ne considero 20 e non una sola?
In principio, esistono infiniti modi
(conformazioni) che permettono di ottenere una
bassa funzione penalità.
Non vi é nessun motivo per sceglierne una
piuttosto che un altra
In linea di principio, la conformazione a piu
bassa funzione penalità é considerabile la
migliore, ma tutte le altre che hanno valore
molto simile sono ugualmente valide
Quindi, preferisco prendere in considerazione un
numero fisso di conformazioni (20, o 30) che
hanno la piu bassa penalità e vedere quanto esse
sono simili tra loro
53
Famiglia di strutture
RMSD
Se le strutture sono molto simili tra loro
significa che tutte le conformazioni che ho
considerato sono molto simili. Avro una
struttura accurata
54
Famiglia di strutture
RMSD
Se le strutture sono molto diverse tra loro
significa che devo considerare come ugualmente
buone conformazioni molto diverse. Avro una
struttura molto poco accurata
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Solution structure of oxidized and reduced
Cytochrome c
Cyt c oxidized
Cyt c reduced
Banci, Bertini, Bren, Gray, Sompornpisut, Turano,
Biochemistry, 1997
Baistrocchi, Banci, Bertini, Turano, Bren, Gray,
Biochemistry, 1996
59
Active site channel of Reduced Monomeric Q133
Copper Zinc Superoxide dismutase
? Reduced Q133M2SOD ? Oxidized human SOD
60
Structure of Ce3 substituted Calbindin D9k
Bertini, Donaire, Jiménez, Luchinat, Parigi,
Piccioli, Poggi, J.Biomol. NMR, 2001,21,85-98
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  • Stucture Calculation
  • Structure Validation
  • Structure Visualisation

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Structure validation
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RMSD
  • How to overlay structures
  • -entire
  • -fragments
  • - bb all heavy atoms

65
RMSD
66
RMSD
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Stereoviews
68
Average pairwise rmsd values calculated for
backbone heavy atoms N, Ca, and C' ("Backbone"),
all heavy atoms ("All heavy"), and backbone heavy
atoms N, Ca, and C' together with heavy atoms of
the best defined side-chains. The values for the
DYANA structures are represented by red bars, and
values for molecular dynamics refined (MDR) and
energy-minimization refined (EMR) structures are
displayed in green and gray, respectively.
Standard deviations are indicated by vertical
error bars.
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Target function analysis
  • Violations lt threshold
  • Energy terms

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PROCHECK
  • The PROCHECK suite of programs provides a
    detailed check on the stereochemistry of a
    protein structure. Its outputs comprise a number
    of plots in PostScript format and a comprehensive
    residue-by-residue listing. These give an
    assessment of both the overall quality of the
    structure, as compared with well-refined
    structures of the same resolution, and also
    highlight regions that may need further
    investigation. The PROCHECK programs are useful
    for assessing the quality not only of protein
    structures in the process of being solved, but
    also of existing structures and those being
    modelled on known structures.

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PROCHECK PROCHECK-NMR
  • The only input required for PROCHECK is the PDB
    file holding the coordinates of the structure of
    interest. For NMR structures, each model in the
    ensemble should be separated by the correct MODEL
    and ENDMDL records

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Model-by-model secondary structures
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Plots of PROCHECK G-factor (all dihedrals) vs.
MOLPROBITY Z-scores (1) calculated for x-ray
crystal structures (circles) deposited in the PDB
during 2000-2004 colored according to resolution
green high-resolution ( 1.8 Å) gray
medium-resolution (1.8 2.5 Å) red
low-resolution (2.5 3.5 Å) and NMR structures
(yellow triangles) deposited in the PDB during
2000-2004 by other leading NMR groups.
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