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.