Protein « photos » with ultrafast lasersProtein « photos » with ultrafast lasers

Goran ZgrablicGoran ZgrablicInstitut de Physique de la Matière CondenséeInstitut de Physique de la Matière Condensée

Université de LausanneUniversité de Lausanne

Summer School of Science, Višnjan Observatory, 27th july 2003

Long organic molecules

which interact in

biological reactions

Proteins: Nano-machines of living cell

3D Protein STRUCTURE:

Protein folding still an unresolved problem

…but to know structure is not enough!

Methods:-X-ray diffraction-NMR (Nuclear Magnetic Resonance) -electron microscopy

“If you want to understand function, study structure”(Francis Crick)

Protein DYNAMICS: the importance of the motion

Molecular interactions:-Hydrophobic-Van der Waals-Electrostatic-Hydrogen bonds

Forces:weak – comparable to thermal motionsMovements: 1 Å (0.1 nm)

Aquaporine in the lipid membrane of the cell

Time window 0.2 ns

Water and protein: a perfect couple

Interactions with environment:Water is essential to biological activity of proteins

Molecular dynamics simulations byB.L. de Groot and H. Grubmüller: Science 294, 2353-2357 (2001))

AquaporineA protein which selectively

passes the water molecules into the cell (red blood cells, kidney, lung,

brain, eye)

Time window 2 ns

“If you want to understand function, study structure”(Francis Crick)

Function= sequence of events over time,

characterised by structural modifications“If you want to understand function, study

time-dependent structures”

Time resolution

Spatial resolution

Conclusion:

Biomolecular structure and dynamics work together to define function

Question:

What do we need to make a movie of the molecules in chemical reaction?

[Fe+III Fe+II(CN)6][Fe+II(CN)6]Fe+III

+

A B AB

400 500 600 700 800

ab

sorp

tio

n

wavelength / nm

INFRAREDUV

SPECTROSCOPY

The color (absorption) distinguishes reactants

from product

SPECTROSCOPY Measuring absorption in time we see progress of

chemical reaction

Time resolution 0.1 s

with shutter camera

Cats are very good physicists!

Question:

What do we need to make a movie of the molecules in chemical reaction?

-> LIGHT PULSE

…but, how short?

The The fundamentalfundamental time scale in time scale in Condensed Matter, Chemistry Condensed Matter, Chemistry and Biologyand Biology

Speed of sound: Speed of sound: 300 m/s-1000m/s => 0.3-1.0 Å in 100 fs300 m/s-1000m/s => 0.3-1.0 Å in 100 fs

•

1fs = 0.00 000 000 000 000 1s = 10-15 s1fs / 1s <=> 1s / 32 million years!

Time scale of half-oscillations:

H2; e = 4155 cm-1 —> 7.6 fs

I2; e = 120 cm-1 —> 270 fs

Ultrafast molecular motionUltrafast molecular motion involved in biological functioninvolved in biological function

Vibrations 20 … 500 fs Torsions 200 fs … 5 psElectron transfer 20 fs … 100 psResonance energy transfer 100 fs…100 ps

Protein folding

10-15 10-12 10-9 10-6 10-3 1 sec

Protein Synthesis

Helix motion

Intermol. charge transferP-P interaction

Intra-molecular motion

Question:

What do we need to make a movie of the molecules in chemical reaction?

-> LIGHT PULSE of few femtoseconds

Femtosecond laser pulsesFemtosecond laser pulses

…but, somebody has

to push the poor cat!

Let’s Use some light

So, we need two fs pulses:

PUMP pulse – photoexcites all the molecules at the same time and starts the chemical reaction

PROBE pulse – measures the absorption change after time we want

t0 = 0 fs

t1 = 100 fst2 = 200 fst3 = 300 fs…

„ „ Femtosecond photography “Femtosecond photography “

Nobel prize in Chemistry 1999:Prof. A. Zewail

“ Femtochemistry “

Schémas von SelmaSchémas von Selma Pulsbreite NOPA’sPulsbreite NOPA’s

-80 -60 -40 -20 0 20 40 60 80

0,0

0,2

0,4

0,6

0,8

1,0

1,2Autocorrelation 640nmPulse width 18.5 fs

Sig

nal (

arb.

uni

ts)

Time(ps)time delay [fs]

distance entre les atomes

éner

gie

0 1 2 3

1

2

3

delay pump - probe / picoseconds

t = 300 fs

Vibration of an isolated molecule

Femtosecond light activated Femtosecond light activated processes in biologyprocesses in biology

some examplessome examples

CHROMOPHORES:CHROMOPHORES:

Molecules that react Molecules that react upon the exposure to upon the exposure to light can be used as light can be used as model systems for the model systems for the study of these ultrafast study of these ultrafast processes processes

Our research focuses on proteins, which can bind CHROMOPHORES (light-sensitive molecules)

Understand molecular physics behind the function

Photosensory proteins

Vision

Photo-taxis

Plant growth

Phytochrome - induction of flowering, chloroplast development, leaf senescence and leaf abscission.

Hemoglobin: dissociation/binding of OHemoglobin: dissociation/binding of O22, CO,..., CO,...

Bacteriorhodopsin: converts light into «food» ATP

Rhodopsin: Rhodopsin: a photosensory protein in eyea photosensory protein in eye

From L. Stryer, Biochemistry

Rhodopsin and the retinal moleculeRhodopsin and the retinal molecule

Retinal chromophore

Cis-trans isomerisationNobel 1961: G. Wald, R.Granit, H.K. Hartline

11-cis all-trans

Femtoseconds and proteins ?Femtoseconds and proteins ?

11-cis all-trans

400 500 600

0.0

0.2

0.4

0.6495 nm

Abs

orpt

ion

(arb

.uni

ts)

Wavelength (nm)400 500 600

0.0

0.2

0.4

0.6548 nm

Abs

orpt

ion

(arb

.uni

ts)

Wavelength (nm)

200 fs

The protein environment controls The protein environment controls at which bond the chromophore will turnat which bond the chromophore will turn

Response to photo-induced charge transfer on chromophore

Protein = has some charges and they can move around

Light can stretch electron cloud when we excite chromophore

Amino acid measures Amino acid measures the induced electric field...the induced electric field...

+ -

……by changing its color from by changing its color from blue to greenblue to green

0

aL

0

ret44444444444444

ret44444444444444

Environment (water) can make a chemical Environment (water) can make a chemical reaction possible, or make it fasterreaction possible, or make it faster

Dynamic Stokes shift – solvent responseProtonated Schiff base in MeOH

Methanol12x10

3

10

8

6

4

2

0

inten

sity

(a. u

.)

700650600550500wavelength [nm]

375 fs 450fs 550fs 725fs 1025fs 1625fs 2725fs

Spectrum changes in time Spectrum changes in time -> water is turning around the molecule-> water is turning around the molecule

Photosynthesis

From http://www.ks.uiuc.edu/Overview/gallery/structure.shtml

Electron transfer in reaction center

Non-radiative energy transfer (Förster)

cytoplasm

periplasm h

Life is made of…

Swiss National Science FoundationRoche Research Foundation “Fondation Herbette” LausanneUni Lausanne

In collaboration with

M. Sheves (Weizmann Institute)

E. Landau (U Texas, Galveston)

J. Heberle & G. Büldt (FZ Jülich)

M. Chergui and his groupM. Chergui and his groupIPMC - Uni Lausanne