seeing enzymes in action with laser t- jump time-resolved xas/xpe/xwas jung y. huang keywords:...
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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? 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.
Why metalloproteins? Why metalloproteins?
Why metalloproteins? 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 ferritinTransport: 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)
Dynamics in Biological Systems 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)
Movements inside Proteins Movements inside Proteins
Many important biochemical processes occur on the time-scales of nanoseconds-microseconds.
Why Laser T-jump? 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.
Why X-ray probes? Why X-ray probes?
• Both e-beam and X-ray can give direct 3D structural information.
• However, sca(hard X ray)=10-3sca (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.
Why X-ray probes? 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.
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 s to 3 s
qS(q)/[EXAFS/XANES] r(r, t)/[abs(,t)] Spatial resolution 0.01A with t=100 ps.
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 J
•Excited size: 50x50 m2
Pulse-to-Pulse Synchronization Timing Scheme
Further Consideration
Estimated Signal Strength:
•For a dilute sample, signal from the excited solutes is about
0.01 of excited solvents. Assuming 10% (depending on abs(exe 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.
Further Consideration
Non Pulse-to-Pulse Based Time-Resolving Technique
ConclusionConclusion
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 highHowever, the reward can be high.