College 7

Een paar van de fysische attributen om biologische processen te begrijpen:

Licht-interakties, modelleren

Interakties met elektromagnetische straling

Peptide α-helix Eiwit

C = koolstofN = stikstofO = zuurstofH = protonR = een aminozuur

Waarom is vibrationele spectroscopie struktuurgevoelig?

X – C – O – H

O – XOω1

ω2

ω1’

ω2’

Het voorbeeld van een diatomisch molekuul

De frequentie van de oscillatie wordt dus bepaald door de veerconstantek en de gereduceerde massa:

ω = (k/m)1/2

Absorptie van licht, ten gevolge van de interaktie tussen het elektromagnetischeveld E(t,w) en het dipoolmoment van het molekuul• Frequentie van het licht moet hetzelfde zijn als ω• Des te groter de puntladingen q, des te groter de interaktie met licht

+q -q

Harmonische beweging, dwz F = -kx

Klassiek: md2x/dt2 = -kx, als we stellen ω2= k/m dan

d2x/dt2 + ω2 x =0

heeft als oplossingen sinus of cosinus funkties van ωt

Protein unfolding

250 -> T -> 360K

Licht absorptie van water en eiwit

Hoe gedraagt water zich, in een eiwit, rond een eiwit, rond een ion, in bulk?

Biological water

Anisotropy decay • fast 200 fs: librational motions• slower decay: molecular jumps, large reorientation

Oa Huib Bakker Amolf

Femtoseconde pump-probe

t=l/c

1 mm => 3 x10-12

s

= 3 ps

Voor en na eenreaktie in een eiwit

Reakties in een eiwit

The pathway for proton transfer in The pathway for proton transfer in Green Fluorescent proteinGreen Fluorescent protein

Proton transfer relay in Green Fluorescent Protein

A B

GFP PhotocycleGFP Photocycle

A-state I-state

Arg96

Kennis, Larsen, Van Stokkum,Vengris, Van Thor, Hellingwerf, Van Grondelle, PNAS 101, 2004

Appearance of greenemission in ~3 and 10 ps,& KIE effect

=> Proton transfer

Global analysis

After averaging, typically 20.000 data points.Analyze time traces at all 256 wavelengths with the same set of exponential decays, and obtain evolution-associated-difference spectra:

A B C

dA(t)/dt = -k1*A(t)

dB(t)/dt = k1*A(t) – k2B(t)

dC(t)/dt = k2*B(t),

with A(0) = 1, B(0)=0 and C(0) = 0

k1 k2

Or more complicated but physicallyrealistic model…..

AWavelength

A B C

S(,t) = Ai()e –t.ki

Visible Pump-Probe and Pump-dump-probe studies: A* decays bi-exponentially into I*. (Chattoraj et al, PNAS 1996; Lossau et al, Chem. Phys. 1996; Kennis et al, PNAS 2004)

GFP Photocycle: important remarksGFP Photocycle: important remarks

Recent calculations suggest that PT starts from end of wire (Vendrell et al JACS 2006 and JACS 2008, Wang et al JPC 2006, PCCP 2007)

FemtoIR studies: protonation of Glu222 occurs with the same kinetics as red shift emission. Therefore, deprotonation of the chromophore was concluded to be the rate limiting step (Stoner-Ma et al, JACS 2005, JPC 2006, van Thor et al JPC 2005)

Multi-pulse control spectroscopy:active manipulation of reactions

Use green pulse to dump I*→I

A* I*

IA

proton transfer

back shuttledum

p pulseexci

tatio

n

3 ps

Kennis, Larsen, Van Stokkum,Vengris, Van Thor, Hellingwerf, Van Grondelle,

PNAS 101, 2004

Femtoseconde pump-probe

t=l/c

1 mm => 3 x10-12

s

= 3 ps

800 nm lightTi:sapphire

oscillator + amplifier

Hurricane (Spectra Physics)

IR1TOPAS(OPA)

MIDIR lightDifference frequency generator

MCT

preamplifierIntegrate&Hold16-bit ADC

1 KHz800 nm

0.8 mJ80-90 fs

350 J

1150-2600 nm

2.4-11m 3 - 1.5 J

200 cm-1 PROBE

SAMPLE

pumpedunpumped

PC

Spectrograph

450 J

400-800 nm~5J, 10-30 fs

PUMP

Visible lightNon-collinear Optical Parametric

Amplifier (second harmonic generator)

Delay30 m = 100 fs

MIR window ~200 cm-1, detect between 1000 and 200 cm-1, excite at 400 nm, 200 nJ.Sample is in moving CaF2 cell, Lissajous scanner,Noise ~10-5 OD in 1 minute

Femtosecond mid-infrared absorptiondifference spectroscopy

OD 3

•Negative: Initial state A

•Positive: New state B

Absorbance

Wavelength

State A

State B

Difference Wavenumbe

r

Why is vibrational spectroscopy structure sensitive?

X – C – O – H

O – XOω1

ω2

ω1’

ω2’

FemtoIR measurementsFemtoIR measurements

Evolution Associated Difference Spectra (EADS) resulting from global analysis

1 2 3 4

Measurements in D2O, excitation@400 nm

X – C – O – H

O

= 1710 cm1

X – C – O–

O

= 1570 cm1

Also checked by site-directed mutagenesis in GFP

FemtoIR measurementsFemtoIR measurements

Evolution Associated Difference Spectra (EADS) resulting from global analysis

1 2 3 4

Measurements in D2O, excitation@400 nm

IR SADS from the parallel modelIR SADS from the parallel model

A

A1*,

I*

I

A2*10; 80ps

3ns

7ns

A

A*I*

I

I0*10ps

80ps

3ns

7ns

(left model)(right model)

Spectral differences between A*1 and A*2 are due to the assumption of early I* formation

Pump-dump-probe spectroscopyPump-dump-probe spectroscopy

• Can we test if the state identified in the infrared is a real intermediate?

• We use pump-dump probe spectroscopy with different pump-dump delays.• Dump delay of 5, 10, 20, 30, 50, 70 and 100 ps have been employed

A

A*

I*

I1

I0*

Gre

en dum

p

Gre

en dump

I0=I2

?

?

Pump-Dump-ProbePump-Dump-Probe

-9 -6 -3 0 5 50 500-70

-60

-50

-40

-30

-20

-10

0

10

mO

D

Time (ps)

508 nm

-9 -6 -3 0 5 50 500-50

-40

-30

-20

-10

0

mO

D

Time (ps)

508 nm

Dump after 5ps

Dump after 100ps

Only one ground state intermediate (I2) is resolved. There is no fast dynamics after the dump pulse is applied

Two ground state intermediates (I1 and I2) are resolved. There is fast dynamics after the dump pulse is applied

Other dump timesOther dump times

The I1 intermediate is resolved only if the dump pulse is applied at least 50 ps after the pump, since on that time scale I* starts to be sufficiently populated to be dumped.

Dump at 70psDump at 15ps

ConclusionsConclusions

We have used ultrafast time resolved infrared and multipulse pump-dump-probe spectroscopy to resolve, with atomic resolution, how, and how fast, protons move through the H-bonding wire in GFP.

All our measurements show that the first event occurring after excitation is the rearrangement of the hydrogen-bonding network of the proton-wire, resulting in the partial protonation of Glu222.

The chromophore releases its phenolic proton only later.

We conclude that the proton transfer events are initiated at the end of the wire.