College 7

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College 7

• Een paar van de fysische attributen om
  biologische processen te begrijpen
• Licht-interakties, modelleren

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Interakties met elektromagnetische straling
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C koolstof N stikstof O zuurstof H
proton R een aminozuur
Peptide
a-helix
Eiwit
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Waarom is vibrationele spectroscopie
struktuurgevoelig?
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-q
q
Het voorbeeld van een diatomisch molekuul
Harmonische beweging, dwz F -kx Klassiek
md2x/dt2 -kx, als we stellen ?2 k/m dan
d2x/dt2 ?2 x 0 heeft als oplossingen sinus
of cosinus funkties van ?t
De frequentie van de oscillatie wordt dus bepaald
door de veerconstantek en de gereduceerde
massa ? (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
  ?
• Des te groter de puntladingen q, des te groter
  de interaktie met licht

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Protein unfolding
250 -gt T -gt 360K
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Licht absorptie van water en eiwit
Hoe gedraagt water zich, in een eiwit, rond een
eiwit, rond een ion, in bulk?
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Biological water

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

Oa Huib Bakker Amolf
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Femtoseconde pump-probe
DtDl/c 1 mm gt 3 x10-12 s 3 ps
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Reakties in een eiwit
Voor en na eenreaktie in een eiwit
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The pathway for proton transfer in Green
Fluorescent protein
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Proton transfer relay in Green Fluorescent
Protein
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GFP Photocycle
Arg96
I-state
A-state
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Kennis, Larsen, Van Stokkum,Vengris, Van Thor,
Hellingwerf, Van Grondelle, PNAS 101, 2004
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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-differenc
e spectra
k1
k2
S(?,t) ? Ai(?)e t.ki
C
B
A
Or more complicated but physicallyrealistic
model..
dA(t)/dt -k1A(t) dB(t)/dt k1A(t)
k2B(t) dC(t)/dt k2B(t), with A(0) 1,
B(0)0 and C(0) 0
Wavelength
?A
A
B
C
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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)

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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
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Kennis, Larsen, Van Stokkum,Vengris, Van Thor,
Hellingwerf, Van Grondelle, PNAS 101, 2004
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Femtoseconde pump-probe
DtDl/c 1 mm gt 3 x10-12 s 3 ps
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Femtosecond mid-infrared absorptiondifference
spectroscopy
800 nm lightTisapphire 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
preamplifier
IntegrateHold 16-bit ADC
pumped
unpumped
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Why is vibrational spectroscopy structure
sensitive?
?2
X C O H
O X
O
?1

• Negative Initial state A
• Positive New state B

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FemtoIR measurements
Evolution Associated Difference Spectra (EADS)
resulting from global analysis
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2
3
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Measurements in D2O, excitation_at_400 nm
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X C O H
X C O
O
O
? 1710 cm?1
? 1570 cm?1
Also checked by site-directed mutagenesis in GFP
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FemtoIR measurements
Evolution Associated Difference Spectra (EADS)
resulting from global analysis
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2
3
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Measurements in D2O, excitation_at_400 nm
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IR SADS from the parallel model
Spectral differences between A1 and A2 are due
to the assumption of early I formation
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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
?
I0I2
A
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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
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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
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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.