Protein « photos » with ultrafast lasers Goran Zgrablic Institut de Physique de la Mati è re Condens é e Universit é de Lausanne Summer School of Science, Višnjan Observatory, 27 th 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 motions Movements:  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 by B.L. de Groot and H. Grubmüller: Science 294, (2001)) Aquaporine A 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 resolutionSpatial 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 +  ABAB

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 fundamental time scale in Condensed Matter, Chemistry and Biology Speed of sound: Speed of sound: 300 m/s-1000m/s => Å in 100 fs 1fs = s = s 1fs / 1s 1s / 32 million years! Time scale of half-oscillations: H 2 ;  e = 4155 cm-1—> 7.6 fs I 2 ;  e = 120 cm-1 —> 270 fs

Ultrafast molecular motion involved in biological function Vibrations 20 … 500 fs Torsions200 fs … 5 ps Electron transfer20 fs … 100 ps Resonance energy transfer100 fs…100 ps Protein folding sec Protein Synthesis Helix motion Intermol. charge transfer P-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 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 t 0 = 0 fs t 1 = 100 fs t 2 = 200 fs t 3 = 300 fs …

„ Femtosecond photography “ Nobel prize in Chemistry 1999: Prof. A. Zewail “ Femtochemistry “

Schémas von Selma Schémas von Selma Pulsbreite NOPA’s Pulsbreite NOPA’s time delay [fs]

distance entre les atomes énergie

 t = 300 fs Vibration of an isolated molecule

Femtosecond light activated processes in biology some examples

CHROMOPHORES: Molecules that react upon the exposure to light can be used as model systems for the study of these ultrafast 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 O 2, CO,...

Bacteriorhodopsin: converts light into «food» ATP

Rhodopsin: a photosensory protein in eye

From L. Stryer, Biochemistry Rhodopsin and the retinal molecule Retinal chromophore Cis-trans isomerisation Nobel 1961: G. Wald, R.Granit, H.K. Hartline 11-cis all-trans

Femtoseconds and proteins ? 11-cis all-trans 200 fs

The protein environment controls at 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 the induced electric field …by changing its color from blue to green

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

Dynamic Stokes shift – solvent response Protonated Schiff base in MeOH

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

Photosynthesis From Electron transfer in reaction center Non-radiative energy transfer (Förster) cytoplasm periplasm h

Life is made of…

Swiss National Science Foundation Roche Research Foundation “Fondation Herbette” Lausanne Uni Lausanne In collaboration with M. Sheves (Weizmann Institute) E. Landau (U Texas, Galveston) J. Heberle & G. Büldt (FZ Jülich) M. Chergui and his group IPMC - Uni Lausanne