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Seeing Enzymes in Action with Laser Tjump 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?
• 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?
• 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)
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
Many important biochemical processes occur on the timescales of nanoseconds-microseconds.
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?
• 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.
Why X-ray probes?
• The local structural methods are beginning to be applied to study
excited-state structures of materials with the use of timeresolved 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 ms to 3 ms
 qDS(q)/[EXAFS/XANES]
 rr(r, t)/[sabs(w,t)]
 Spatial resolution 0.01A with Dt=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 mJ
•Excited size: 50x50 mm2
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
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.
Further Consideration
Non Pulse-to-Pulse Based Time-Resolving Technique
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.