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College 5

College 5. 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 5

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  1. College 5 Een paar van de fysische attributen om biologische processen te begrijpen: Licht-interakties, modelleren

  2. Interakties met elektromagnetische straling

  3. C = koolstof N = stikstof O = zuurstof H = proton R = een aminozuur Peptide α-helix Eiwit

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

  5. -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

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

  7. Femtoseconde pump-probe Dt=Dl/c 1 mm => 3 x10-12 s = 3 ps

  8. Reakties in een eiwit Voor en na eenreaktie in een eiwit

  9. The pathway for proton transfer in Green Fluorescent protein

  10. A B Proton transfer relay in Green Fluorescent Protein

  11. Femtoseconde pump-probe Dt=Dl/c 1 mm => 3 x10-12 s = 3 ps

  12. 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

  13. 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

  14. FemtoIR measurements Evolution Associated Difference Spectra (EADS) resulting from global analysis 1 2 3 4 Measurements in D2O, excitation@400 nm

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

  16. FemtoIR measurements Evolution Associated Difference Spectra (EADS) resulting from global analysis 1 2 3 4 Measurements in D2O, excitation@400 nm

  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: 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

  18. (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

  19. 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

  20. 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

  21. 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

  22. 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.

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