One of the most fundamental questions in biology, chemistry, and physics is how the molecules interact, so called, reaction mechanisms. Our laboratory strives to understand the reaction mechanisms in molecular detail in gas phase, solution phase, and solid phase spanning amorphous phase, polycrystalline and single crystals. Traditionally, femtosecond spectroscopy has been used to achieve this goal, however, spectroscopic data, in most cases, fail to provide direct information on the structural changes such as bond lengths and bond angles. To remedy this, we combine the traditional femtoscience with direct structural tools such as diffraction (scattering and crystallography), EXAFS, and NMR. These techniques can be applied to a wide range of systems, encompassing small molecules, nano-scale complexes, and macromolecules such as polymers, proteins and DNA. A typical experiment is conducted in a pump-probe manner; an optical pulse such as femtosecond laser pulse is directed to the sample of interest to initiate a desired reaction, and after a well-defined time delay, a probing pulse such as an ultrashort x-ray pulse is sent to the sample undergoing a reaction. Then, the signal resulted from the interaction of the reacting system and the probing pulse captures the molecular actions in real time. Since the measured signal (in this case, diffraction signal) is a function of molecular structures, the time-dependant data at various time delays contains a clue to the molecular reaction mechanisms and a novel data analysis of the time-resolved signal finally reveals the mechanism.
Molecular Dynamics of Small Molecules, Nano-Scale Molecules and Macromolecules in Solution: Time-Resolved X-ray Liquidography (Solution Scattering)
Third-generation synchrotron radiation facility such as European Synchrotron Radiation Facility (ESRF) and Advanced Photon Source (APS) can generate an X-ray pulse as short as 100 picoseconds. With the advent of X-ray free electron lasers (XFELs) such as PAL-XFEL, SACLA and LCLS, even femtosecond X-ray pulses are now available, opening up a new possibility of capturing the movement of molecules in real time. Our laboratory conducts time-resolved experiments by utilizing such ultrashort X-ray pulses as a probe. Of great interest is the molecular structure of short-lived intermediates and solute-solvent interactions in solution phase. X-ray has a much longer penetration depth than electron does, and this characteristic makes X-ray suited for solution studies. A series of successful time-resolved X-ray diffraction experiments have been conducted on reactions of various small molecules and macromolecules such as nano-scale complexes and proteins.
Time-Resolved Spectroscopy: Femtosecond Transient Absorption Spectroscopy, 2-Dimensional Electronic Coherent Spectroscopy, Nanosecond Transient Absorption Spectrosocpy and Nanosecond Transient Grating Spectroscopy
Along with time-resolved X-ray/electron diffraction, our laboratory conducts a variety of time-resolved optical spectroscopy experiments such as femtosecond transient absorption spectroscopy, 2-dimensional electronic coherent spectroscopy, nanosecond transient absorption spectroscopy and nanosecond transient grating spectroscopy. Porous materials such as COFs and MOFs as well as small molecules and proteins have been studied by utilizing these tools.
Protein Structural Dynamics: Time-Resolved X-ray Crystallography
Our laboratory is interested in the reaction mechanisms of various proteins such as a blue light photoreceptor, bacteriorhodopsin, phytochrome, and LOV2 domain. These proteins play indispensible roles in the signal transduction pathway of a cellular system. The time-resolved X-ray Laue diffraction on a single crystal is currently the only method which can provide us detailed real-time structural information in sub-nanosecond time resolution at room temperature.The experiment is conducted at a third generation synchrotron facility such as ESRF and APS as the time-resolved X-ray liquid experiment. The difference is that the long-range order found in a single crystal enhances the diffraction signal and reduce the background significantly. Diffuse signal from disordered molecules becomes sharp peaks. Generally, a ns or fs laser pulse triggers the protein dynamics in a single crystal of proteins and a polychromatic, ultrashort X-ray pulse from a beamline of a synchrotron radiation facility sweeps through the single crystal. The resulting diffraction pattern is then recorded in a charge-coupled device (CCD) based detection system. Typically a better time resolution can be achieved when the single crystal is kept still and a polychromatic X-ray beam is used for diffraction unlike a traditional X-ray crystallographic experiment where a diffraction pattern of a monochromatic X-ray beam is measured on an oscillating crystal. However, our laboratory is also developing a new technique of achieving a better time resolution with the traditional monochromatic X-ray pulses so that a wider range of protein reactions can be studied.
Ab-Initio and DFT Quantum Chemical Calculation and Molecular Dynamics Simulation
Modern scientific advances have confirmed that experiments alone cannot provide a full story and convincing explanations on complex reaction mechanisms. Therefore, all our experimental endeavors accompany theoretical calculations including both ab-initio and DFT quantum chemical calculations and molecular dynamics simulations. Selected examples are molecular structures of short-lived intermediates, the photo-reaction of solute molecules in solution, and reaction pathway of proteins.
Time-Resolved Electron Diffraction
With the advance of fs laser technology and electron gun design, the time resolution of ultrafast electron diffraction has reached near 1 ps. This impressive time resolution has enabled us to capture the molecular structure of short-lived intermediates. However, the fundamental bond-making and bond-breaking processes occur in femtosecond regime, which is beyond the current technology. Our laboratory is developing a next-generation ultrafast electron diffraction technique, which is femtosecond electron diffraction (FED). This new technique is based on a light-driven RF electron gun producing a near-relativistic electron pulse. Upon realization of this new technique, a molecular vibration will be directly captured, fulfilling one of chemists' dreams.
Direct observation of bond formation in solution with femtosecond X-ray liquidographyNature 518 (2015) 385; J. Phys. B: At. Mol. Opt. Phys. 48 (2015) 244005; Struct. Dyn. 3 (2016) 043209
Professor Ihee and his research group visualized the real-time progress of bond formation in a gold trimer complex, [Au(CN)2]3–, with atomic resolution using femtosecond X-ray solution scattering at an X-ray free electron laser (XFEL). The making and breaking of chemical bonds are essential processes in chemical reactions, and the ultrafast dynamics of bond breaking processes have been well studied using time-resolved techniques. However, it is extremely difficult to study the dynamics of bond making because the bond making is a bimolecular process and thus is limited by slow diffusion in solution phase. This work was the first example of directly observing the structural dynamics of bond formation process in solution phase and demonstrated the full power of femtosecond X-ray solution scattering equipped with femtosecond time resolution and atomic-level structural resolution.
Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallographyNat. Chem. 5 (2013) 212; Nat. Chem. 6 (2014) 259
Professor Ihee and his research group revealed the atomic-level structural changes involved in the signaling process of photoactive yellow protein using time-resolved X-ray crystallography. From time-resolved X-ray crystallography measurement of the isomerization process in photoactive yellow protein, the Ihee group revealed that the early stage of the isomerization on the nanosecond time scale proceeds via bifurcated pathways with different chromophore structures. This result was highly valued because it provided the atomic-level structural change of the chromophore, which cannot be probed by time-resolved spectroscopy, with the progress of the protein structural transition.
Determining molecular structures of reaction intermediates in solution: Capturing the bridged structure of C2H4IScience 309 (2005) 1223; J. Phys. Chem. A 109 (2005) 10451; J. Chem. Phys. 124 (2006) 124504
Professor Ihee and his research group have used time-resolved X-ray Liquidography (TRXL) to identify the intermediates of the elimination reaction of haloethane (C2H4I2). Upon irradiation at 267 nm, haloethane dissociates into a haloethyl radical and an iodine atom. Of particular interest is the molecular structure of the haloethyl radical because a bridged structure rather than open classical structure (anti and gauche) had been proposed to explain the observed stereoselectivity of certain chemical processes but had never been directly observed. According to the TRXL result, the bridged structure is strongly favored rather than open classical structure and is also predicted by quantum mechanical calculation.
Protein structural dynamics in solution revealed by pump-probe X-ray protein liquidography and time-resolved X-ray Laue crystallography; Hemoglobin, Myoglobin, and Photoactive yellow proteinPNAS 102 (2005) 7145; Nat. Methods 5 (2008) 881; J. Phys. Chem.B 113 (2009) 13133; Chem. Commun. 47 (2010) 289; J. Phys. Chem. Lett. 2 (2011) 350
Professor Ihee demonstrates tracking of protein structural changes with pump-probe X-ray protein liquidography with picosecond time resolution. We investigated the tertiary and quaternary conformational changes of hemoglobin and myoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. By providing insights into the structural dynamics of proteins functioning in their natural environment, pump-probe X-ray protein liquidography complements and extends results obtained with time-resolved optical spectroscopy and X-ray Laue crystallography. We also identify a complex chemical mechanism and all atomic structures of five distinct structural intermediates of the blue light photoreceptor photoactive yellow protein (PYP) using time-resolved X-ray Laue crystallography.
Revealing an unknown reaction pathway of photocatalyst; Ru3(CO)12Angew. Chem. Int. Ed. 47 (2008) 5550; J. Am. Chem. Soc. 132 (2010) 2600
TRXL was used to probe the photolysis of Ru3(CO)12 in cyclohexane, and a new intermediate (Ru3(CO)10 with terminal CO only) was identified besides the two μ-CO intermediates known from ultrafast IR spectroscopy. Three intermediates, Ru3(CO)10 with terminal CO only, Ru3(CO)11(μ-CO) and Ru3(CO)10(μ-CO), undergo different photodissociation pathways at 260 nm and 390 nm.
The reaction mechanism of metathesis probed by time-dependent fluorescence quenching; Metal-based catalystsJ. Am. Chem. Soc. 130 (2008) 16506; J. Am. Chem. Soc. 132 (2008) 12027
Quantitative catalyst-substrate association relationships of diverse metathesis Mo and Ru catalysts to their substrates are determined directly by a general method based on FRET principle. The determined substrate preferences exhibit the order of alkyne > alkene > allene for Schrock or Schrock-Hoveyda Mo catalysts, allene > alkene > alkyne for Grubbs or Crubbs-Hoveyda 1st generation Ru catalysts, and alkyne > allene > alkene for Grubbs or Grubbs-Hoveyda 2nd generation Ru catalysts.
"Light-induced protein structural dynamics in bacteriophytochrome revealed by time-resolved x-ray solution scattering", Sci. Adv., 2022, 8, eabm6278. [Full text] [Supporting Information]
"Solvent-modulated proton-coupled electron transfer in an iridium complex with an ESIPT ligand", Chem. Sci., 2022, 13, 3809-3818. [Full text] [Supporting Information]
"Determining the charge distribution and the direction of bond cleavage with femtosecond anisotropic x-ray liquidography", Nat. Commun., 2022, 13, 522. [Full text] [Supporting Information]
"Optical Kerr Effect of Liquid Acetonitrile Probed by Femtosecond Time-Resolved X-ray Liquidography", J. Am. Chem. Soc., 2021, 143, 14261-14273. [Full text] [Supporting Information]
"Filming ultrafast roaming-mediated isomerization of bismuth triiodide in solution", Nat. Commun., 2021, 12, 4732. [Full text] [Supporting Information]
"Uncovering the Conformational Distribution of a Small Protein with Nanoparticle-Aided Cryo-Electron Microscopy Sampling", J. Phys. Chem. Lett., 2021, 12, 6565-6573. [Full Text] [Supporting Information]
"Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering", Nat. Commun., 2021, 12, 3677. [Full Text] [Supporting Information] [Full-text access (SharedIt)] [Highlighted in the Editors’ Highlights]
"Effect of the abolition of intersubunit salt bridges on allosteric protein structural dynamics", Chem. Sci., 2021, 12, 8207-8217. [Full Text] [Supporting Information]
"Femtosecond X-ray Liquidography Visualizes Wavepacket Trajectories in Multidimensional Nuclear Coordinates for a Bimolecular Reaction", Acc. Chem. Res., 2021, 54, 1685-1698. [Full Text] [Supporting Information]
"Ultrafast structural dynamics of in-cage isomerization of diiodomethane in solution", Chem. Sci., 2021, 12, 2114-2120. [Full Text] [Supporting Information]
"Enhancement of Energy Transfer Efficiency with Structural Control of Multichromophore Light‐Harvesting Assembly", Adv. Sci., 2020, 7, 2001623. [Full Text] [Supporting Information]
"Mapping the emergence of molecular vibrations mediating bond formation", Nature, 2020, 582, 520-524. [Full Text] [Supporting Information] [Full-text access (SharedIt)] [Covered in C&EN]
"Protein folding from heterogeneous unfolded state revealed by time-resolved X-ray solution scattering", Proc. Natl. Acad. Sci., 2020, 117, 14996-15005. [Full Text] [Supporting Information][Highlighted in Commentary of Proc. Natl. Acad. Sci.] [Spotlighted in ESRF Highlights 2020]
"Ultrafast Charge Transfer Coupled with Lattice Phonons in Two-dimensional Covalent Organic Frameworks", Nat. Commun., 2019, 10, 1873. [Full Text] [Supporting Information]
“Structural Dynamics of Bismuth Triiodide in Solution Triggered by Photoinduced Ligand-to-Metal Charge Transfer”, J. Phys. Chem. Lett., 2019, 10, 1279-1285. [Full Text] [Supporting Information]
“Direct Observation of a Transiently Formed Isomer During Iodoform Photolysis in Solution by Time-Resolved X-ray Liquidography”, J. Phys. Chem. Lett., 2018, 9, 647-653. [Full Text] [Supporting Information] [Erratum]
“Ultrafast X-ray crystallography and liquidography”, Annu. Rev. Phys. Chem., 2017, 68, 673-697. [Full Text]
“Atomistic characterization of the active-site solvation dynamics of a model photocatalyst”, Nat. Commun., 2016, 7, 13678. [Full Text][Supporting Information]
“Lanthanum-catalysed synthesis of microporous 3D graphene-like carbons in a zeolite template”, Nature, 2016, 535, 131-135. [Full Text][Supporting Information]
“Tracking reaction dynamics in solution by pump-probe X-ray absorption spectroscopy and X-ray liquidography (solution scattering)”, Chem. Commun., 2016, 52, 3734-3749. [Full Text]
“Direct observation of bond formation in solution with femtosecond X-ray scattering”, Nature, 2015, 518, 385-389. [Full Text][Supporting Information]
“Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis”, J. Phys. Chem. Lett., 2015, 6, 5045-5050. [Full Text]
“Protein Structural Dynamics Revealed by Time-Resolved X-ray Solution Scattering”, Acc. Chem. Res., 2015, 48, 2200-2208 (Review Article). [Full Text][Highlighted in the Front Cover]
“Single-Step Fabrication of Quantum Funnels via Centrifugal Colloidal Casting of nanoparticle Films”, Nat. Commun., 2015, 6, 7772. [Full Text][Supporting Information]
“A Photoresponsive Smart Covalent Organic Framework", Angew. Chem. Int. Ed., 2015, 127, 8828-8831. [Full Text][Supporting Information]
“Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser”, Nat. Commun., 2015, 6, 6772. [Full Text][Supporting Information]
“Random-Graft Polymer-Directed Synthesis of Inorganic Mesostructures with Ultrathin Frameworks”, Angew. Chem. Int. Ed., 2014, 53, 5117-5121. [Full Text][Supporting Information]
“Solvent-Dependent Molecular Structure of Ionic Species Directly Measured by Ultrafast X-Ray Solution Scattering”, Phys. Rev. Lett., 2013, 110, 165505. [Full Text][Supporting Information]
“Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography”, Nat. Chem., 2013, 5, 212-220. [Full Text][Supporting Information]
“Filming the Birth of Molecules and Accompanying Solvent Rearrangement”, J. Am. Chem. Soc., 2013, 135, 3255-3261. [Full Text][Supporting Information]
“Conjugated organic framework with three-dimensionally ordered stable structure and delocalized π clouds”, Nat. Commun., 2013, 4, 2736. [Full Text][Supporting Information]
“Microtubes with Rectangular Cross-Section by Self-Assembly of a Short b-Peptide Foldamer”, J. Am. Chem. Soc., 2012, 134, 20573-20576. [Full Text][Supporting Information]
“Direct observation of cooperative protein structural dynamics of homodimeric hemoglobin from 100 picoseconds to 10 milliseconds with pump-probe X-ray solution scattering”, J. Am. Chem. Soc., 2012, 134, 7001-7008. [Full Text][Supporting Information]
“Protein structural dynamics of photoactive yellow protein in solution revealed by pump-probe X-ray solution scattering”, J. Am. Chem. Soc., 2012, 134, 3145-3153. [Full Text][Supporting Information]
“The short lived signalling state of the photoactive yellow protein photoreceptor revealed by combined structural probes”, J. Am. Chem. Soc., 2011, 133, 9395-9404. [Full Text][Supporting Information]
“Anisotropic Picosecond X-ray Solution Scattering from Photoselectively Aligned Protein Molecules”, J. Phys. Chem. Lett., 2011, 2, 350-352. [Full Text]
“Ultrafast X-ray Solution Scattering Reveals Different Reaction Pathways in the Photolysis of Triruthenium Dodecacarbonyl (Ru3(CO)12) after Ultraviolet and Visible Excitation”, J. Am. Chem. Soc., 2010, 132, 2600-2607. [Full Text][Supporting Information]
“Visualizing Solution-Phase Reaction Dynamics with Time-Resolved X-ray Liquidography”, Acc. Chem. Res., 2009, 42, 356-366 (Review Article). [Full Text]
“The Initial Catalyst-Substrate Association Step in the Enyne Metathesis Catalyzed by Grubbs Ruthenium Complex Probed by Time-Dependent Fluorescence Quenching”, J. Am. Chem. Soc., 2008, 130, 16506-16507. [Full Text][Supporting Information]
“Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering”, Nat. Methods, 2008, 5, 881-887 (Highlighted as a front cover article). [Full Text][Supporting Information][Highlighed in the front Cover]
“Ultrafast X-ray solution scattering reveals a new reaction intermediate in the photolysis of Ru3(CO)12”, Angew. Chem. Int. Ed., 2008, 47, 5550-5553. [Full Text][Supporting Information]
“Capturing Transient Structures in the Elimination Reaction of Haloalkane in Solution by Transient X-ray Diffraction”, J. Am. Chem. Soc., 2008, 130, 5834-5835. [Full Text]
“Transient X-ray Diffraction Reveals Global and Major Reaction Pathways for Iodoform in Solution”, Angew. Chem. Int. Ed., 2008, 47, 1047-1050. [Full Text][Supporting Information]
“Photochemical Reaction Pathways of Carbon Tetrabromide in Solution Probed by Picosecond X-ray Diffraction”, J. Am. Chem. Soc., 2007, 129, 13584-13591. [Full Text][Supporting Information]
“Spatiotemporal reaction kinetics of an ultrafast photoreaction pathway visualized by time-resolved liquid x-ray diffraction”, Proc. Natl. Acad. Sci. USA, 2006, 103, 9410-9415. [Full Text][Supporting Information]
“Ultrafast X-ray diffraction of transient molecular structures in solution”, Science, 2005, 309, 1223-1227. [Full Text][PDF][Perspectives] [Perspectives-PDF][Supporting Information]
“Visualizing Reaction Pathway in Photoactive Yellow Protein from Nanoseconds to Seconds", Proc. Natl. Acad. Sci. 2005, 101, 7147-7150. [Full Text]