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Seeing Enzymes in Action with Laser T-jump Time-resolved XAS/XPE/XWAS

Seeing Enzymes in Action with Laser T-jump Time-resolved XAS/XPE/XWAS. Jung Y. Huang http://www.jyhuang.idv.tw/. Keywords: liquid phase, metalloproteins, Laser T-jump, X-ray probe, pulse-to-pulse synchronization. Why study liquid-phase reactions?.

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Seeing Enzymes in Action with Laser T-jump Time-resolved XAS/XPE/XWAS

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  1. Seeing Enzymes in Action with Laser T-jump Time-resolved XAS/XPE/XWAS Jung Y. Huang http://www.jyhuang.idv.tw/ Keywords: liquid phase, metalloproteins, Laser T-jump, X-ray probe, pulse-to-pulse synchronization

  2. Why study liquid-phase reactions? • Majority of biological cellular processes and industrial applications occur in liquid phases. Water is a major contributor to a protein's 3-D structure and in reverse the protein also controls the structuring of its surrounding water.

  3. Why metalloproteins?

  4. Why metalloproteins? • It is estimated that about 1/4-1/3 of all proteins requires metals to carry out their functions. Metal ions involved are usually coordinated by nitrogen, oxygen or sulfur atoms belonging to amino acid residues of the protein. • Metalloproteins play many different functional roles in cells, such as Storage: iron storage protein ferritin Transport: Oxygen transport proteins myoglobin and hemoglobin; Electron-transfer vectors for redox reaction such as Cytochromes (Fe), Plastocyanin (Cu), Chlorophyll-containing proteins (Mg) Enzymes:Hexokinase (Mg), methionine synthase (Co), Carbonic anhydrase (Zn), Superoxide dismutase (Cu), Nitrogenase (Mo) Signal Transduction:Calmodulin (Ca) Regulation:Transcription factors (Zn)

  5. Dynamics in Biological Systems • Protein structure and stability; folding/unfolding • Protein Function • Protein reaction kinetics Biological activity correlated with dynamic transition of structure (http://www.jyhuang.idv.tw/SingleMoleculeBiophysics.aspx)

  6. Movements inside Proteins Many important biochemical processes occur on the time-scales of nanoseconds-microseconds.

  7. Why Laser T-jump? • The introduction of pulsed lasers excitation as triggers of the biochemical processes brought dramatic improvement in the experimental time resolution. However, this methodology is inapplicable to molecules without suitable chromophores. • Laser T-jump methodology has evolved into one of the most versatile and generally applicable methods for studying fast biomolecular kinetics.

  8. Why X-ray probes? • Both e-beam and X-ray can give direct 3D structural information. • However, ssca(hard X ray)=10-3ssca (e). Electron beam cannot penetrate deeply into the bulk of a sample, thus it is limited to surface and gas-phase study. • For condensed phase study, such as in liquid phase, several advantages can be yielded from X-ray probing technique, such as XAS, XPS, XRD, etc.

  9. Why X-ray probes? • The local structural methods are beginning to be applied to study excited-state structures of materials with the use of time-resolved pump-probe experiments.

  10. Laser T-jump Time-resolved XAS/XPE/XWAS • Target: Direct structural characterization of short-lived intermediates. • Approach: Signal from delayed X-ray pulse probes the change in the electronic and spatial correlation function. • Data Acquisition Procedure: Collect time-resolved X-ray scattering/absorption/emission data from -3 ms to 3 ms qDS(q)/[EXAFS/XANES] rr(r, t)/[sabs(w,t)]  Spatial resolution 0.01A with Dt=100 ps.

  11. Pulse-to-Pulse Synchronization Timing Scheme Characteristics of the excitation laser: • Pulse Repetition Rate (PRR): 347 kHz; 1/2 of the PRR of storage ring • Pulse Energy: 4 mJ • Excited size: 50x50 mm2

  12. Pulse-to-Pulse Synchronization Timing Scheme

  13. Further Consideration Estimated Signal Strength: • For a dilute sample, signal from the excited solutes is about0.01 of excited solvents. Assuming 10% (depending on sabs(wexe) and the focused laser intensity) optical excitation efficiency, S/BKG<10-3. How to improve the sensitivity? • Use the chemical selectivity of XRA to distinguish the signal from excited solutes from the background signal.

  14. Further Consideration

  15. Non Pulse-to-Pulse Based Time-Resolving Technique

  16. Conclusion To have a successful trXAS program for dynamic study of catalysts and proteins, we need a strong and coherent strategy for combining input from multiple experimental methods and theory (MD and models for structural retrieving). However, the reward can be high.

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