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College 7. Een paar van de fysische attributen om biologische processen te begrijpen: Licht-interakties, modelleren. Interakties met elektromagnetische straling. C = koolstof N = stikstof O = zuurstof H = proton R = een aminozuur. Peptide. α - helix. Eiwit. ω 2 ’. ω 2. X – C – O – H.

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college 7

College 7

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

Licht-interakties, modelleren

slide3

C = koolstof

N = stikstof

O = zuurstof

H = proton

R = een aminozuur

Peptide

α-helix

Eiwit

slide4

ω2’

ω2

X – C – O – H

O – X

O

ω1

ω1’

Waarom is vibrationele spectroscopie struktuurgevoelig?

slide5

-q

+q

Het voorbeeld van een diatomisch molekuul

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

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
protein unfolding
Protein unfolding

250 -> T -> 360K

slide8

Licht absorptie van water en eiwit

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

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

Oa Huib Bakker Amolf

slide10

Femtoseconde pump-probe

Dt=Dl/c

1 mm => 3 x10-12 s

= 3 ps

slide11

Reakties in een eiwit

Voor en na eenreaktie in een eiwit

slide13

A

B

Proton transfer relay in Green Fluorescent Protein

slide14

GFP Photocycle

Arg96

I-state

A-state

slide15

Appearance of greenemission in ~3 and 10 ps,

& KIE effect

=> Proton transfer

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

global analysis
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:

k1

k2

S(,t) =  Ai()e –t.ki

C

B

A

Or more complicated but physicallyrealistic model…..

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

Wavelength

A

B

A

C

slide18

GFP Photocycle: important remarks

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)

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)

  • 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)
slide19

Multi-pulse control spectroscopy:

active manipulation of reactions

Use green pulse to dump I*→I

proton transfer

A*

I*

3 ps

excitation

dump pulse

I

A

back shuttle

slide21

Femtoseconde pump-probe

Dt=Dl/c

1 mm => 3 x10-12 s

= 3 ps

slide22

OD 3

Femtosecond mid-infrared absorptiondifference spectroscopy

800 nm lightTi:sapphire

oscillator + amplifier

Hurricane (Spectra Physics)

Visible lightNon-collinear Optical Parametric

Amplifier

(second harmonic generator)

1 KHz

800 nm

0.8 mJ

80-90 fs

350 mJ

Delay

30 mm = 100 fs

400-800 nm

~5mJ, 10-30 fs

1150-2600 nm

IR1TOPAS

(OPA)

MIDIR lightDifference

frequency

generator

450 mJ

2.4-11mm

3 - 1.5 mJ

D200 cm-1

PROBE

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

PUMP

Spectrograph

SAMPLE

MCT

PC

Integrate&Hold

16-bit ADC

preamplifier

pumped

unpumped

slide23

ω2’

Absorbance

State B

State A

Wavelength

ω1’

Difference

Wavenumber

Why is vibrational spectroscopy

structure sensitive?

ω2

X – C – O – H

O – X

O

ω1

  • Negative: Initial state A
  • Positive: New state B
slide24

FemtoIR measurements

Evolution Associated Difference Spectra (EADS) resulting from global analysis

1

2

3

4

Measurements in D2O, excitation@400 nm

slide25

X – C – O – H

X – C – O–

O

O

= 1710 cm1

= 1570 cm1

Also checked by site-directed mutagenesis in GFP

slide26

FemtoIR measurements

Evolution Associated Difference Spectra (EADS) resulting from global analysis

1

2

3

4

Measurements in D2O, excitation@400 nm

slide27

(left model)

(right model)

A1*,

A2*

A*

10ps

10; 80ps

I0*

80ps

I*

I*

3ns

3ns

7ns

I

7ns

I

A

A

IR SADS from the parallel model

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

pump dump probe spectroscopy
Pump-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*

I0*

I*

?

Green dump

Green dump

I1

?

I0=I2

A

slide29

Pump-Dump-Probe

Dump after 5ps

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

Dump after 100ps

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

slide30

Other 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 15ps

Dump at 70ps

slide31

Conclusions

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.