College 7 Een paar van de fysische attributen om biologische processen te begrijpen: Licht-interakties, modelleren.

Presentatie over: "College 7 Een paar van de fysische attributen om biologische processen te begrijpen: Licht-interakties, modelleren."— Transcript van de presentatie:

1 College 7 Een paar van de fysische attributen om biologische processen te begrijpen: Licht-interakties, modelleren

2 Interakties met elektromagnetische straling

3 Peptideα-helixEiwit C = koolstof N = stikstof O = zuurstof H = proton R = een aminozuur

4 Waarom is vibrationele spectroscopie struktuurgevoelig? X – C – O – H O – X O ω1ω1 ω2ω2 ω1’ω1’ ω2’ω2’

5 Het voorbeeld van een diatomisch molekuul De frequentie van de oscillatie wordt dus bepaald door de veerconstante k en de gereduceerde massa: ω = (k/m) 1/2 Absorptie van licht, ten gevolge van de interaktie tussen het elektromagnetische veld 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: md 2 x/dt 2 = -kx, als we stellen ω 2 = k/m dan d 2 x/dt 2 + ω 2 x =0 heeft als oplossingen sinus of cosinus funkties van ωt

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7 Protein unfolding 250 -> T -> 360K

8 Licht absorptie van water en eiwit Hoe gedraagt water zich, in een eiwit, rond een eiwit, rond een ion, in bulk?

9 Biological water Anisotropy decay fast 200 fs: librational motions slower decay: molecular jumps, large reorientation Oa Huib Bakker Amolf

10 Femtoseconde pump-probe  t=  l/c 1 mm => 3 x10 -12 s = 3 ps

11 Voor en na een reaktie in een eiwit Reakties in een eiwit

12 The pathway for proton transfer in Green Fluorescent protein

13 Proton transfer relay in Green Fluorescent Protein AB

14 GFP Photocycle A-state I-state Arg96

15 Kennis, Larsen, Van Stokkum,Vengris, Van Thor, Hellingwerf, Van Grondelle, PNAS 101, 2004 Appearance of green emission in ~3 and 10 ps, & KIE effect => Proton transfer

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17 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 = -k 1 *A(t) dB(t)/dt = k 1 *A(t) – k 2 B(t) dC(t)/dt = k 2 *B(t), with A(0) = 1, B(0)=0 and C(0) = 0 k1k1 k2k2 Or more complicated but physically realistic model….. AA Wavelength A B C S( ,t) =  Ai(  )e – t.k i

18 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 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)

19 Multi-pulse control spectroscopy: active manipulation of reactions Use green pulse to dump I* → I A* I* I A proton transfer back shuttle dump pulse excitation 3 ps

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

21 Femtoseconde pump-probe  t=  l/c 1 mm => 3 x10 -12 s = 3 ps

22 800 nm light Ti:sapphire oscillator + amplifier Hurricane (Spectra Physics ) IR1 TOPAS (OPA) MIDIR light Difference frequency generator MCT preamplifier Integrate&Hold 16-bit ADC 1 KHz 800 nm 0.8 mJ 80-90 fs 350  J 1150-2600 nm 2.4-11  m 3 - 1.5  J   200 cm -1 PROBE SAMPLE pumpedunpumped PC Spectrograph 450  J 400-800 nm ~5  J, 10-30 fs PUMP Visible light Non-collinear Optical Parametric Amplifier (second harmonic generator) Delay 30  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 CaF 2 cell, Lissajous scanner, Noise ~10 -5 OD in 1 minute Femtosecond mid-infrared absorption difference spectroscopy OD 3

23 Negative: Initial state A Positive: New state B Absorbance Wavelength State A State B Difference Wavenumber Why is vibrational spectroscopy structure sensitive? X – C – O – H O – X O ω1ω1 ω2ω2 ω1’ω1’ ω2’ω2’

24 FemtoIR measurements Evolution Associated Difference Spectra (EADS) resulting from global analysis 1234 Measurements in D 2 O, excitation@400 nm

25 X – C – O – H O  = 1710 cm  1 X – C – O – O  = 1570 cm  1 Also checked by site-directed mutagenesis in GFP

26 FemtoIR measurements Evolution Associated Difference Spectra (EADS) resulting from global analysis 1234 Measurements in D 2 O, excitation@400 nm

27 IR SADS from the parallel model A A1*, I* I A2* 10; 80ps 3ns 7ns A A* I* I I0*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

28 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 A* I* I1I1 I0*I0* Green dump I 0 =I 2 ? ?

29 Pump-Dump-Probe Dump after 5ps Dump after 100ps Only one ground state intermediate (I 2 ) is resolved. There is no fast dynamics after the dump pulse is applied Two ground state intermediates (I 1 and I 2 ) are resolved. There is fast dynamics after the dump pulse is applied

30 Other dump times The I 1 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 70ps Dump at 15ps

31 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.