NiPS 2010

I neutroni rivelano la relazione tra dinamica molecolare e funzione

biologica in proteine, membrane e cellule, in vivo

Giuseppe (Joe) Zaccai

Institut Laue-Langevin, GrenobleE-mail: zaccai@ill.fr

NiPS 2010

> 60 000 strutture nella PDB

NiPS 2010

Capire una struttura è capire le forze che la mantengano

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Haloarcula marismortuiSale ! Archaeoglobus fulgidus

Temperatura !

Malate dehydrogenase

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_H +. _S +∆H+ ∆S+

∆H+,-,0 ∆S +,-,0

Privalov and Khechinashvili (1974) J. Mol. Biol., 86, 665-684

Calorimetria

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BPTI (Go et al., 1983)

3.56 ps-1

0.21 ps-1

Normal M

odes

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r i (0)

€

S(Q,ω)= 12π

I(Q∫ ,t)exp(−iωt)dt

I(Q,t)= 1N

<eiQ.rk(t)

k, j

∑ e-iQ.rj (0) >

r i (t )

Neutron Incoherent Scattering

Single Particle Scattering

k0

k1 Q

σ (H) >> σ (D and other nuclei)

NiPS 2010

≈ ( 1 /  Q )  2   Å  2

≈ 2 Å  2

on IN13 at ILL

time ≈ 100 pson the backscatteringspectrometer IN13

≈ 25  Å  2

H  2  O diffusing

H on protein

The elast ic scan : a window in length – t ime

NiPS 2010

T ( K )0 40 80 120 160 200 240 280

0.00

0.30

0.60

0.90

1.20

k' = 0.3N/m

k = 2N/m

k = 3N/m

< u

2>

Å2(

)

320

1.50

Data from Doster et al. (1989), Cordone et al. (2000)

hydrated powder

in trehalose glass

Neutron Scattering:Mean square displacements and

Effective Force Constants in Myoglobin

€

<k'>=2kB

d u2

dT

−1

< u2 > and < k’ > are experimental parameters~ flexibility,

~ resilience respectively

T’

IN 13 8 microeV 100 ps

Parametri

« High th

roughput !

»

NiPS 2010

Lavori sul Lisozima di Paciaroni e colleghi:

La dinamica di una proteina dipendefortemente dall’ambiente acquoso !

NiPS 2010Frauenfelder et al. (1988)

Una chiave per capire :

“Energy landscapes

or seascapes”

NiPS 2010

∆G

~ kBT ’ < k >

< k ’ >

quasi-harmonic approximation

√< u2 >

∆G

d

√< u2 >

< k ’ > ~ ∆G / 2d2

Bicout and Zaccai, Biophys. J., 2001Ea

√< u2 >

d

€

<k'>~1/d

dTRate×∆τ×d2

€

< k '>~ 1/d

dTRate × ∆τ × d 2

Rate ~ A exp(− E a

RT)

1/ < k '> ~ 0 ~ E a

RT >> 1 or << 1

< k '> ~ constant ~ E a

RT ~ 2 to 1

Ea

d

Bicout and Zaccai, Biophys. J., 2001

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Vita colorata del sale

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1.0

1.5

2.0

2.5

3.0

3.5

4.0

270 280 290 300 310 320 330

0 10 20 30 40 50

Temperature (K)

<u2

> (Å

2 )

(C)

2M NaCl H2 O

2M NaCl D2 O

2M KCl D2 O

< k’ > = 0.1 N/m

< k’ > = 0.2 N/m

< k’ > = 0.5 N/m

200 mg/ml protein

Fluctuations and force constants depend on the solventand correlate with stabil i ty

Tehei et al.(2001)

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

20 30 40 50 60 70 80 90

300 310 320 330 340 350 360

Aggregation

Temperature (C)

Norm

ali

zed

Ell

ipti

city

222 n

m

(K)

2M NaCl H2 O

2M NaCl D2 O2M KCl D2 O

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There are

Dynamics-Function-Activity

Relations

Dynamics – Function-Activity

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Biological Dynamics is not as simple as 'Flexibil i ty is required for activity ' - i t is Function Specif ic !

NiPS 2010Gabel et al., Biophys J, 2009

Human BuChE and Soman

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Hb e temperatura fisiologica

Hb in …

solution

powder

Stadler et al., Biophys J, 2009Stadler et al., Biophys J, 2008

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Purple Membrane

H. Salinarum

Bacteriorhodopsin

CP

CP

EC

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Photocycle

specif ic f lexibil i ty

K

J

LN

O

BR570

M412

hν

ECH+

M412

CP

H+CP

EC

AG

F

E DC

B

H+

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Dry PM

Wet PM

Harmonic domain

Dynamical transition

Activation of photocyle !

H+

‘transition of methyls’

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Labelled sample :

In-vivo labelling:Fully deuterated PM with hydrogenated retinal, Trp, Met.

Retinal binding pocketExtracellular moiety of BR

Labelled BR: Sample preparation:

EC

CP

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2.5

2.0

1.5

1.0

0.5

0.0

300200100

Temp. [K]

B

2.5

2.0

1.5

1.0

0.5

0.0

30025020015010050

Temp. [K]

D

2.5

2.0

1.5

1.0

0.5

0.0

300200100

Temp. [K]

A

2.5

2.0

1.5

1.0

0.5

0.0

30025020015010050

Temp. [K]

C

Large amplitude motions in labelled ↔ unlabelled PM

0 % r.h. 75 % r.h.

86 % r.h. 93 % r.h.

< u2 >, < k’ >Separate f lexibi l i ty and

r igidity

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<u2 >

[Å

2 ]

<u2 >

[Å

2 ]

Lipids BR

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Less soft core = valve

H+

Soft body

Softer lipid environment

All are hydration ...

and temperature dependent

Degrees of ‘Softness’

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Dynamic adaptation to heat

LDH of Rabbit

MDH of Methanocaldococcus

jannaschii

0

20

40

60

80

100

120

0 20 40 60 80 100

Opt

imal

Act

ivit

y (%

)

T(°C)

Tehei, M., D. Madern, B. Franzett i & G. Zaccai, J Biol Chem (2005)

0

20

40

60

80

100

120

0 20 40 60 80 100 120

T(° C)

B

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0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

270 280 290 300 310 320 330

<u2

> (

Å2)

T(K)

Soft Rabbit muscle LDH Hard Methanocaldococcus jannaschii MalDH

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Escherichia colimesophile 37°C

Aquaspir i l lum arct icumpsychrophile 4°C

Proteus mirabi l ismesophile 37°C

Thermus thermophilusthermophile 65°C

Aquifex pyrof i lushyperthermophile 85°CTehei et al. (2004)

Adaptation of the Proteome

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0.5

1.0

1.5

2.0

2.5

3.0

270 280 290 300 310 320

0 10 20 30 40<

u2 >

(Å2 )

T (K)

T (° C)

E. coli : al ive and dead

NiPS 2010

1.0

1.5

2.0

2.5

270 280 290 300 310 320

0 10 20 30 40

<u

2 > (

Å2 )

T(° C)

T (K)

<k'>=(0,21±0,03) N/m A

1,0

1,5

2,0

2,5

270 280 290 300 310 320

0 10 20 30 40

<k'>=(0,39±0,01) N/m

<u2

> (

Å2)

T (K)

B

T (° C)

1.0

1.5

2.0

2.5

270 280 290 300 310 320

0 10 20 30 40

T (K)

<u2

> (

Å2)

<k'>=(0,67±0,11) N/m C

T (° C)

1.0

1.5

2.0

2.5

270 280 290 300 310 320

0 10 20 30 40

<k'>=(0,60±0,01) N/m<

u2 >

(Å2 )

T (K)

DT (° C)

P. mirabi l isA. arct icum

A. pyrofi lusT. thermophilus

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0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8<

k'

> (

N/m

)

Psychrophile Mesophile Thermo / Hyperthermophile

The effective force constant increases to maintain stabil i ty

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0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6¦(

< u

2 >)

(Å

)

Psychrophile Mesophile Thermo / Hyperthermophile

Same rms f luctuation at physiological temperature !

√

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Una costante di forza adatta per mantere lo stesso valore della

f lutuazione alla temperatura f isiologica

L’evoluzione ha scelto la dinamica !

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

¦(<

u2 >

) (

Å)

Psychrophile Mesophile Thermo / Hyperthermophile

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< u2

> (Å

2 )

Heparan Sulfate, a cell surface polysaccharide

Jasnin et al., PCCP, 2010

Cartografia dinamica della cellula

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< u2

> (Å

2 )

Proteine di membrana Lipidi poliSaccaridi <u

2 > [

Å2 ]

1.00

P

L

S

NiPS 2010

Michel Ferrand, Valérie Réat, Uschi Lehnert, Martin Weik, Moeava Tehei, Frank Gabel, Katy Wood, Marion Jasnin, Andreas Stadler…

BacteriorhodopsinDieter Oesterhelt and his Lab, MPI Martinsried

Proteins and CellsDominique Madern et Bruno Franzetti, IBS, Grenoble, Margaret et Ben Zion Ginzburg,

Jérusalem, Martine Moulin et Michael Haertlein, D-LAB, Marie-Thérèse Giudici-Orticoni, Marseille

Red Blood CellsGerhard Artmann and his Lab in Aachen, Georg Bueldt in Juelich

Senza chi …

NiPS 2010

Grazie !