Presentazione di PowerPoint

1
Struttura e dinamica di proteine e
metalloproteine in soluzione
Claudio Luchinat CERM - University of
Florence Fabio Arnesano University of Bari
2
What is addressed in 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 2002 352
3
Evolution of NMR
Magnetic field strengths
NMR experiments dimensions
900
800
750
4D
600
500
3D
360
2D
270
220
200
1D
100
60
40
30
4
Structural biology by NMR in Florence
Metalloprotein structures solved ca. 15
iron-sulfur proteins ca. 30 heme-proteins ca. 50
copper zinc calcium proteins
Since 2001 CERM is a partner of the SPINE project
(Structural Proteomics IN Europe)
Claudio Luchinat, XX IUCr 2005
5
NMR Spectroscopy
It deals with nuclear spins in a magnetic
field NMR spectroscopy detects the interaction
between a magnetic field and the magnetic moments
of the nuclear spins in the sample
mI
mI
mI magnetic moment
I angular momentum
I(I1)
A spinning charge with spin angular momentum I
gives rise to a magnetic moment. In a magnetic
field its axis of rotation precesses around the
direction of the field with a frequency called
Larmor frequency w-gB0
I
B0
mI
w
6
The NMR transition
It occurs between two nuclear spin levels
at 800 MHz (18.8 Tesla)
B0
P1 (mI½)
mI½
P0 (mI-½)
Low Sensitivity
nuclear Zeeman Hamiltonian
7
The NMR transition
The NMR experiment detects bulk quantities
When an ensemble of spins is considered, the spin
magnetic moments sum up to give a macroscopic
magnetization, M, along the magnetic field axis
(conventionally z)
A large collection of spins
At equilibrium Mz M Mx My 0
z
Mz
y
x
8
The NMR Experiment
To have a spin transition, a magnetic field B1 ,
oscillating in the range of radiofrequencies and
perpendicular to z, is applied (perturbing pulse)
After the pulse is switched off, the
magnetization precesses in the xy plane and
relaxes to equilibrium
The B1 field creates coherence among the spins
(they all have the same phase) and net
magnetization in the x,y plane is created
The current induced in a coil by the
magnetization precessing in the xy plane is
recorded. It is called FID.
9
The NMR spectrum
FID
The Fourier Transform of the FID provides the NMR
spectrum
Spectrum
½ Fmax(w)
wI
w1
w
10
The NMR spectrum
B0
n
nI
n2
High Resolution
Two nuclei with DDE gt Dn1/2 give two separate NMR
signals
At 800 MHz, for a molecule of 10kDa ? average
1H Dn1/2 10-9 cm-1
11
NMR parameters
Chemical shifts - Determined by the effective
magnetic field sensed by the resonating nucleus
Relaxation rates - Depend on the nature of the
resonating spin, on the interaction energy with
the surrounding, and on the motions of the
lattice
J couplings - Depend on the nature of the coupled
spins, on the number of bonds and on the bond
angles
12
Multidimensional NMR
If a series of pulses are applied and more than
one time interval is present, Fourier
transformations over all the various time
dimensions provide multiple frequency dimensions
Preparation
Evolution
Mixing
Detection
t2
t1
tm
13
Multidimensional NMR
If a series of FIDs (all with the same t2 range)
are measured with different t1 values, Fourier
transformation along t2 provides
t1
w2
wI
wJ
Then, Fourier transformation along t1 provides
Peaks at frequencies w2 wI and w1 wJ (plus
the symmetric) originate from coupling between
the two spins
14
Chemical Shift
Beff (1-?) Bo
Bo
? e- shielding
?B0
?ppm (? - ?REF) x106 / ?REF
Since the chemical shift of a nucleus is
sensitive to the environment, it should also
contain structural information. The chemical
shift index is a method for predictingprotein
secondary structures from the observed chemical
shift of a-1H , a/b-13C and carbonyl 13C
Chemical shift index plotted for the first 65
residues of interleukin 4, R. Powers et al.
Biochemistry 31,4334 (1992)
15
Relaxation mechanisms

• The main interactions causing fluctuating
• magnetic fields, and consequently relaxation,
  are
• dipolar
• CSA
• quadrupolar
• chemical exchange
• paramagnetic couplings

16
Dipolar interaction
Molecular tumbling generates fluctuating magnetic
fields at the nucleus which provide a relaxation
mechanism
Bo
J
q
q
r
I
I
r
J
17
Two spin system
The two spins I and J with quantum number ½ are
dipolarly coupled
bb
W1I
W2
ab
W1J
W0
W1J
ba
W1I
aa
W transition probability per unit time
I,J
18
The definition of NOE
Fractional variation of signal intensity when
another signal is perturbed
Transient (equivalent to NOESY exps)
Steady state
I
I
J
J
Reference
Irradiated
Difference
Theoretical basis laid by Overhauser, Phys. Rev.
91, 476 (1953)
19
The equations of NOE
For two spins I and J dipole-dipole coupled the
longitudinal transfer of magnetization is
described by
B0
J
I
q
rI,J
20

The definition of NOE
Transient
Steady state
-1
h
Steady state
-0.5
Transient
0
t
0.2
0.4
0.6
21
From the analysis of NOESY experiment
1H
1H
All 1H within 6Å from the amide-1H signal
intensity ? 1H-1H distance (NOE)
22
Distance constraints
NOESY volumes are proportional to the inverse of
the sixth power of the interproton distance and
to the correlation time for the dipolar coupling
Upon rotational averaging
B0
mJ
mI
q
r
23
Dihedral angle constraints
Dihedral angles are related to 3J coupling
constants through the Karplus equation
H
?
Ha
N
Ca
Experimentally determined
JHNHa gt 8Hz -155lt fy120 lt -85
JHNHa lt 4.5Hz -70lt fy120 lt -30
4.5HzltJHNHalt 8Hz f,y values are determined
from JHNC
Taken from crystallographic data
24
Contributions to 1JNH

• The 1JNH is the sum of three components
• 1JNH (BO) 1JNH (0) JNHdip (BO) ?DFS (BO)

-dDFS values depend on the rotational regime of
the molecule -In the case of axially symmetric
rotational diffusion, it depends on the relative
orientations of the diffusion and CSA tensors and
on the orientation of the NH bond vector with
respect to the two tensors
Tjandra, Grzesiek and Bax, JACS 1997
25
Field and orientation dependence of 1JNH
Axial tensor (case Dcrh 0)
J(800)-J(500)lt0
J(800)-J(500)gt0
?zz
?zz
1H
?
?
1H
15N
15N
Residual dipolar coupling
26
Oblate Spheroid
Prolate Spheroid
Semi-axes a, b, b (a gt b)
a, b, b (a lt b)
27
Cross correlation effects
Mackor EL, MacLean C, J. Chem. Phys. (1966)
28
Cross correlation effects
Mackor EL, MacLean C, J. Chem. Phys. (1966)
29
Protein mobility, timescales and NMR
tc
Global reorientation
r
Water exchange
conformational exchanges
Motions of sidechains
Folding/unfolding
fast librations
log(time/s)
NH
ND
MD simulations
Exchange rates
Heteronuclear NOE, T1 and T2 relaxation
CPMG and T1? experiments
NMRD measurements