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Metodi per la determinazione
della struttura di biomolecole
Che informazioni offre la struttura?
•
•
•
•
•
•
Conformazione dei siti attivi e di legame
Orientazione dei residui conservati
Interpretazione di meccanismi
Visualizzazione di cavità
Calcolo di potenziale elettrostatico
…
The paper
The model
Maurice Wilkins
M.H.F. Wilkins, A.R. Stokes, H.R. Wilson:
Molecular Structure of Deoxypentose Nucleic
Acids. Nature 171, 738 (1953)
Rosalind Franklin
R.E. Franklin and R.G. Gosling
Molecular Configuration in Sodium
Thymonucleate, Nature 171, 740 (1953)
Nobel laureates 1962
Wilkins, Perutz, Crick, Steinbeck, Watson, Kendrew
Cristallografia a raggi X
• Ottenere cristalli della proteina
– 0.3-1.0 mm
– Le singole molecole sono ordinate in modo
periodico, ripetitivo.
• La struttura è determinata dai dati di
diffrazione.
Diffrazione di raggi X
Schmid, M. Trends in Microbiology, 10:s27-s31.
X-ray emission
Instrumentation
or synchrotron X-rays
Instrumentation
SR beam
Instrumentation
ESRF - Grenoble
Instrumentation
Injection process
Instrumentation
SR tunnel
Instrumentation
Focusing magnet
Instrumentation
Front - end
Instrumentation
Beamline
Single Crystal X-ray Diffraction Basics
The Bragg law
l = 2 d sinq
sinq/l = 1/(2 d)
d = l / (2 sinq)
Single Crystal X-ray Diffraction Basics
The Bragg law
l = 2 d sinq
sinq/l = 1/(2 d)
d = l / (2 sinq)
Single Crystal X-ray Diffraction Basics
The Bragg law
l = 2 d sinq
sinq/l = 1/(2 d)
d = l / (2 sinq)
Cristallografia a raggi X
• Le proteine devono cristallizzare
– Grande quantità
– Solubili
• Accesso a radiazione adatta
• Tempo di calcolo per risolvere la struttura
Forza Ionica
Esercizio: calcolare la differenza di forza ionica tra le soluzioni:
0.01 M NaCl
0.01 M CaCl2
0.01 M CaSO4
Used for crystallization experiments
Le solubilità della proteina è
determinata dalle interazioni soluto
solvente dovute alle cariche
superficiali della proteina
Lys, Arg, Asp, Glu
L’aumento della forza ionica
della soluzione favorisce la
interazione tra le cariche
superficiali della proteina ed il
solvente polare
Un eccesso di forza ionica della
soluzione favorisce le
solvatazione degli ioni stessi a
scapito della solvatazione della
proteina. Il sale compete con la
proteina e la solvatazione
diminuisce all’aumentare della
forza ionica
Crystal growth
Crystal growth
Crystal growth
Supersaturation
Nucleation
+ growth
Supersolubility curve
Solubility curve
Growth only
Unsaturation
Protein concentration
Risoluzione
Campo magnetico
NMR
NOE (Nuclear Overhauser Effect)
Il campo magnetico
Magnete a 900 MHz
Magnete a 400 MHz
Costo commerciale di uno spettrometro a 900 MHz: ca. 5 M€
n. di spettrometri 900 MHz operativi al mondo < 10
The Magnet
History
First magnets were built using
ferromagnetic material=
permanent magnet
Then Electromagnets:
i.e. field was generated by
wiring of conducting material
Now: cyomagnets: i.e. electromagnets
made of superconducting wire.
A “cutted” magnet
L’energia della E= -m•B
0
transizione NMR
DE=(h/2p)B0

NUCLEUS
m=-1/2
DE=h0
• Sensitivity
E
m=+1/2
B0
B0 MAGNETIC
FIELD
Larmor Frequency
I due livelli energetici sono
Il campo magnetico B0 serve per creare la
degeneri se B0=0
separazione di energia tra i 2 livelli
L’energia della E= -m•B
0
transizione NMR
DE=(h/2p)B0
Devo applicare una
radiofrequenza alla frequenza
DE=h0
Per stimolare la transizione!
m=-1/2
DE=h0
E
m=+1/2
B0
Il campo di radiofrequenza
utilizzato per eccitare la
transizione
si chiama anche B1
Larmor Frequency
B1 deve emettere una radiofrequenza in corrispondenza della frequenza di Larmor
Some paradigmatic examples
Why?
NMR is a unique method to obtain information in solution
AT THE ATOMIC LEVEL.
Each individual atom has a peculiar resonance frequency.
Because the resonance frequency depends on the environment of
the atom, EACH atom has a different resonance frequency and
can, therefore be identified
CH3-CH2-OH
equivalent
Sensibilità dell’Esperimento
NMR
S/N ≈ N 5/2 B3/2
N = Numero di spins che contribuiscono al segnale
 rapporto giromagnetico del nuclide studiato
B Camp magnetico utilizizzato
NMR Spectrum to 3D Structure
H
H
Interactions
Spectrum
H
H
H H H
H
H
H
H
Structure
Challenges For Determining
Protein Structures Using NMR
• Proteins have thousands of signals
• Assign the specific signal for each atom
• Thousands of interactions between atomsalso need to be assigned
• Need to transform from NMR spectrum
through interpretation of scalar and dipolar
interactions to generate 3D coordinates
Resonance Assignment
CH3-CH2-OH
OH
CH2 CH3
Which signal from which H atoms?
The key attribute: use the scalar and dipolar couplings to
match the set of signals with the molecular structure
Proteins Have Many Signals
1H
NMR Spectrum of Ubiquitin
~500 resonances
A large number of signals are overlapped
A Critical Feature of
Protein NMR Spectra
• Only some nuclei are coupled
Each amino acid gives rise to an independent NMR
sub-spectrum, which is much simpler than the
complete protein spectrum
Methods have been
developed to extract
each sub-spectrum
from the whole
Basic Strategy to Assign
Resonances in a Protein
1. Identify resonances for each amino
acid
T
L
S
G
S
R
G
2. Put amino acids in order
- Sequential assignment (R-G-S,T-L-G-S)
- Sequence-specific assignment
1
2
3
4
5
6
7
R-G-S-T-L-G-S
Critical Features of
Protein NMR Spectra
• The nuclei are not all mutually coupled
• Regions of the spectrum correspond to
different parts of the amino acid
• Tertiary structure leads to increased
dispersion of resonances
Regions of the 1H NMR Spectrum
are Further Dispersed by the 3D Fold
What would the unfolded protein look like?
Proteins Have Overlapped
Signals
1H
NMR Spectrum of Ubiquitin
Resolve resonances by multi-dimensional experiments
Resolve Peaks By Multi-D NMR
A BONUSregions in
2D spectra provide
protein fingerprints
If 2D cross peaks
overlap go to 3D
or 4D …..
Solution to the Protein
Challenge
1. Increase dimensionality of spectra to
better resolve signals: 1234
2. Detect signals from heteronuclei
(13C,15N)
t2
t1
t3
Heteronuclear nD NMR
1. Increase dimensionality of spectra to
better resolve signals: 1234
2. Detect signals from heteronuclei (13C,15N)
Better resolution of signals/chemical shifts not
correlated between nuclei
More information to identify signals
Lower sensitivity to MW of protein
Structure Determination by NMR
NMR Experimental
Observables Providing
Structural Information
• Backbone conformation from chemical
shifts (Chemical Shift Index- CSI)
• Distance constraints from NOEs
• Hydrogen bond constraints
• Backbone and side chain dihedral angle
constraints from scalar couplings
• Orientation constraints from residual
dipolar couplings
1H-1H
Distances From NOEs
Long-range
(tertiary structure)
Sequential
Intraresidue
A
B
C
D
••••
Z
Medium-range
(helices)
Challenge is to assign all peaks in NOESY spectra
Determining Protein Fold
Before Structure
Calculations
1. Determine secondary structure
•CSI directly from assignments
•Medium-range NOEs
2. Add key long-range NOEs to fold
Approaches to Identifying NOEs
• 1H-1H (homonuclear)
2D
3D
•
15N-
or 13C-dispersed
3D
(heteronuclear)
4D
1H
1H
1H
1H
1H
Identifying Unique NOEs
• Filtered, edited NOE:
based on selection of
NOEs from two
molecules with unique
labeling patterns.
Labeled
protein
Unlabeled
peptide
Only NOEs at the interface
• Transferred NOE:
H
H
based on: 1) faster build-up
H
kon
of NOEs in large versus
small molecules; 2) signal
koff
H
of free state when in excess
and exchanging quickly
Only NOEs from bound state
Hydrogen Bonds
C=O
H-N
• NH chemical shift to low field
• Slow rate of NH exchange with solvent
• Characteristic pattern of NOEs
• Scalar couplings across the H-bond
When H-bonding atoms are known 
can impose a series of distance/angle
constraints to enforce standard H-bond
geometries
Angles From Scalar (J)
Couplings
 Must accommodate multiple solutions multiple J values
Orientational Constraints
From Dipolar (D) Couplings
Ho
Reports angle of internuclear vector relative
to magnetic field Ho
F2
F3
F1
 Must accommodate multiple solutions multiple orientations
NMR Structure Calculations
• Objective is to determine all conformations
consistent with the experimental observables
• In contrast to X-ray crystallography, NMR
observes the atoms indirectly
• NMR data is not perfect: noise, incomplete
Multiple solutions: caused by
uncertainties
in the experimental constraints
Conformational Ensemble
Representing an NMR
Structure
C
N
Precision: RMSD of the ensemble
NMR Structure Calculations:
Two Primary Approaches
1. Start with polymer and fold
Distance Geometry
Hybrid Simulated Annealing
2. Start with all conformations and restrict
Systematic Grid Search
Filters
NMR Structures: The
Challenges
• Distances alone are not sufficient
• Interiors good, but surfaces can be
poor because of few distances/high
dynamics- variable resolution
• Poor electrostatics
– Simplified treatment (scaled charges)
– No explicit solvent, dielectric
Variable Resolution
Interiors well
defined, surfaces
more variable
Regular
secondary
structures well
defined, loops
more variable
Backbone and
side chain trends
are similar
Stereopairs
Linewidth is Dependent on MW
A
B
A
15N
B
Linewidth
determined by
size of particle
15N
15N
Fragments
have narrower
linewidths
1H
1H
1H
Dynamics/Constraints
Regions with higher
flexibility will exhibit
fewer H-H NOEs
Dynamics/Uncertainty in
Structures

Weak correlation
Strong correlation 


High uncertainty
MAY be correlated
with a high degree
of flexibility
Risonanza Magnetica Nucleare
(NMR)
•
•
•
•
•
•
Proteine in soluzione
Limite di dimensione ~ 40 kDa
Proteine stabili a lungo
Marcatura con 15N, 13C, 2H.
Strumentazione molto costosa
Tempo per assegnare le risonanze
Pro e contro
X-ray
NMR
•
Richiede cristalli, problematico
•
•
Non ha limiti (teorici) di
grandezza
Possibile in soluzione, più
semplice
•
Limitato a proteine fino a circa
300 residui
•
Piú preciso
•
Meno preciso
•
Risoluzione
•
Numero di vincoli
•
Struttura può essere deformata
dai cristalli, rigida
•
Struttura nativa in soluzione,
flessibile
•
Una “soluzione“
•
Molti modelli
X-ray
NMR
Protein Data Bank (PDB)
•
URL: http://www.rcsb.org/pdb/
•
Coordinate 3-D di strutture proteiche
•
Formato unico
•
Tutte le strutture risolte con i raggi X e NMR
•
Più vecchia della maggior parte degli altri database
•
Strutturata male a causa dello sviluppo storico
Il Protein Data Bank
Crescita del PDB
Motivi strutturali depositati ogni anno
Percentuale di nuovi motivi strutturali
Formato PDB I
HEADER
COMPND
COMPND
SOURCE
SOURCE
AUTHOR
AUTHOR
...
REMARK
REMARK
REMARK
REMARK
REMARK
REMARK
REMARK
REMARK
REMARK
...
SEQRES
SEQRES
SEQRES
...
ONCOGENE PROTEIN
06-JUN-91
121P
H-RAS P21 PROTEIN COMPLEX WITH GUANOSINE-5'-[B,G-METHYLENE]
2 TRIPHOSPHATE
HUMAN (HOMO SAPIENS) CELLULAR HARVEY-RAS GENE TRUNCATED AND
2 EXPRESSED IN (ESCHERICHIA COLI)
U.KRENGEL,K.SCHEFFZEK,A.SCHERER,W.KABSCH,A.WITTINGHOFER,
2 E.F.PAI
121P
121P
121P
121P
121P
121P
121P
2
3
4
5
6
7
8
1
1 REFERENCE 1
1 AUTH
U.KRENGEL,I.SCHLICHTING,A.SCHEIDIG,M.FRECH,J.JOHN,
1 AUTH 2 A.LAUTWEIN,F.WITTINGHOFER,W.KABSCH,E.F.PAI
1 TITL
THE THREE-DIMENSIONAL STRUCTURE OF P21 IN THE
1 TITL 2 CATALYTICALLY ACTIVE CONFORMATION AND ANALYSIS OF
1 TITL 3 ONCOGENIC MUTANTS
1 REF
NATO ASI SER.,SER.A
V. 220
183 1991
1 REFN
ASTM NALSDJ US ISSN 0161-0449
2002
121P
121P
121P
121P
121P
121P
121P
121P
121P
17
18
19
20
21
22
23
24
25
1
2
3
121P
121P
121P
56
57
58
166
166
166
MET THR GLU TYR LYS LEU VAL VAL VAL GLY ALA GLY GLY
VAL GLY LYS SER ALA LEU THR ILE GLN LEU ILE GLN ASN
HIS PHE VAL ASP GLU TYR ASP PRO THR ILE GLU ASP SER
Formato PDB II
HELIX
HELIX
...
SHEET
SHEET
SHEET
...
TURN
TURN
TURN
...
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
...
HETATM
HETATM
HETATM
HETATM
...
1 A1
2 A2
LYS
SER
1 S
2 S
3 S
6 GLU
6 GLU
6 THR
1 T1
2 T2
3 T3
ALA
ILE
ALA
1
2
3
4
5
6
7
8
9
10
11
1324
1325
1326
1327
16
65
GLN
THR
37
49
2
11
46
83
25
74
ILE
THR
VAL
VAL
GLU
ASN
1
1
46 0
58 -1
9 1
O
N
LEU
LEU
53
6
N
O
LYS
ASP
42
54
14
49
86
121P
121P
80
81
121P
121P
121P
85
86
87
121P
121P
121P
91
92
93
104
105
106
107
108
109
110
111
112
113
114
N
CA
C
O
CB
CG
SD
CE
N
CA
C
MET
MET
MET
MET
MET
MET
MET
MET
THR
THR
THR
1
1
1
1
1
1
1
1
2
2
2
-7.176
-5.913
-5.903
-6.703
-4.712
-4.594
-3.193
-4.325
-4.966
-4.759
-4.312
32.630
31.928
30.860
30.881
32.869
33.420
34.558
35.886
29.934
28.930
29.597
-6.655
-6.676
-5.600
-4.654
-6.415
-4.990
-4.899
-4.618
-5.760
-4.751
-3.441
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
14.06
17.27
16.41
16.12
17.94
19.41
21.82
23.68
15.08
16.71
16.63
121P
121P
121P
121P
121P
121P
121P
121P
121P
121P
121P
PG
O1G
O2G
O3G
GTO
GTO
GTO
GTO
167
167
167
167
5.150
4.768
4.164
4.834
32.173
32.597
32.683
30.641
22.030
23.390
21.069
22.025
1.00
1.00
1.00
1.00
11.69
13.29
12.61
13.18
121P1427
121P1428
121P1429
121P1430
X
Y
Z
B-factor
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
ATOM
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
CA
C
O
N
CA
C
O
CB
OG
N
CA
C
O
CB
OG1
CG2
GLU
GLU
GLU
SER
SER
SER
SER
SER
SER
THR
THR
THR
THR
THR
THR
THR
225
225
225
226
226
226
226
226
226
227
227
227
227
227
227
227
-0.900
-0.185
-0.514
0.788
1.534
2.231
1.883
2.572
3.237
3.242
3.989
4.274
4.179
5.354
5.114
6.256
-1.002
0.146
1.329
-0.203
0.805
1.806
1.952
0.130
-0.941
2.478
3.417
2.705
3.296
3.797
4.682
4.492
39.233
39.970
39.758
40.823
41.594
40.681
39.514
42.515
41.848
41.223
40.410
39.080
38.022
41.074
42.172
40.065
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
70.00
70.00
70.00
70.00
70.00
68.89
70.00
70.00
70.00
65.51
70.00
56.25
44.63
70.00
70.00
70.00
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
1HXN
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185