College 7

1. College 7 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

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 Dt=Dl/c 1 mm => 3 x10-12 s = 3 ps

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

12. The pathway for proton transfer in Green Fluorescent protein

13. A B Proton transfer relay in Green Fluorescent Protein

14. GFP Photocycle Arg96 I-state A-state

15. 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

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. 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)

19. 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

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

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

22. 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

23. 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

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

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

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

27. (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

28. 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

29. 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

30. 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

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.