Jungkweon Choi

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

Role of Water in Directing Diphenylalanine Assembly into Nanotubes and Nanowires

[*] Prof. H. Ihee, J. Kim, Dr. J. Choi Center for Time-Resolved Diffraction, Department of Chemistry Graduate School of Nanoscience & Technology (WCU), KAIST 335 Gwahangno, Yuseong-gu Daejeon, 305-701 (Republic of Korea) E-mail: hyotcherl.ihee@kaist.ac.kr Prof. S. O. Kim, T. H. Han, J. S. Park Department of Materials Science and Engineering (BK21) KAIST Institute for the Nanocentury, KAIST 335 Gwahangno, Yuseong-gu Daejeon, 305-701 (Republic of Korea) E-mail: sangouk.kim@kaist.ac.kr Dr. Y. Kim Korea Research Institute of Standards and Science P.O. Box 102, Yuseong-gu Daejeon, 305-340 (Republic of Korea)

pH-Induced Intramolecular Folding Dynamics of i-Motif DNA

Using the combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) technique, we investigate the mechanism and dynamics of the pH-induced conformational change of i-motif DNA in the bulk phases and at the single-molecule level. Despite numerous studies on i-motif that is formed from cytosine (C)-rich strand at slightly acidic pH, its detailed conformational dynamics have been rarely reported. Using the FRET technique to provide valuable information on the structure of biomolecules such as a protein and DNA, we clearly show that the partially folded species as well as the single-stranded structure coexist at neutral pH, supporting that the partially folded species may exist substantially in vivo and play an important role in a process of gene expression. By measuring the FCS curves of i-motif, we observed the gradual decrease of the diffusion coefficient of i-motif with increasing pH. The quantitative analysis of FCS curves supports that the gradual decrease of diffusion coefficient (D) associated with the conformational change of i-motif is not only due to the change in the intermolecular interaction between i-motif and solvent accompanied by the increase of pH but also due to the change of the shape of DNA. Furthermore, FCS analysis showed that the intrachain contact formation and dissociation for i-motif are 5-10 times faster than that for the open form. The fast dynamics of i-motif with a compact tetraplex is due to the intrinsic conformational changes at the fluorescent site including the motion of alkyl chain connecting the dye to DNA, whereas the slow intrachain contact formation observed from the open form is due to the DNA motion corresponding to an early stage interaction in the folding process of the unstructured open form.

Protein Structural Dynamics of Photoactive Yellow Protein in Solution Revealed by Pump–Probe X-ray Solution Scattering

Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.

Transient X‐ray Diffraction Reveals Global and Major Reaction Pathways for the Photolysis of Iodoform in Solution

The understanding of reaction mechanisms requires detailed information about the processes that take place during the reactions. Various time-resolved optical spectroscopic tools have been developed to track such processes, and reaction dynamics can now be routinely investigated, with a time resolution of tens of femtoseconds, by using these optical techniques. Typical information provided by timeresolved spectroscopy includes the time constants of reaction intermediates and limited structural information. In most cases, however, detailed structural information, such as the bond lengths and angles in reaction intermediates, are extremely difficult to obtain from time-resolved optical spectroscopy (except for a few favorable cases in which they can be deduced from time-resolved vibrational spectroscopy and multi-dimensional spectroscopy measurements). Replacing the optical probe pulses in time-resolved spectroscopy measurements with either electron or Xray pulses converts the optical resonances in energy space into atomic interferences in real space, which offers a complementary—and more direct—way to investigate the structural dynamics of molecular reactions. Because of the relatively low penetration depth of electrons, X-rays are more suitable for probing crystalline and liquid samples; for example, all the atoms in a protein can be tracked during its biological function by means of timeresolved X-ray crystallography, but to do so it is necessary to produce single protein crystals. This limitation has been recently overcome by introducing transient X-ray liquidography (TXL), a method through which the transient molecular structures present in a liquid sample can be captured in one dimension by performing time-resolved Xray diffraction measurements in the liquid phase. TXL is a new technique that can be used to investigate reactions in solution, where most of the chemically and biologically relevant processes take place. Since the diffraction patterns generated by the short X-ray pulses arriving at the sample after laser excitation include all the molecular structures present in the irradiated volume, the analysis of TXL data can reveal the structural evolution of all the reaction pathways in the sample (limited only by the signal-to-noise ratio of the diffracted difference signal). We recently succeeded in studying the structural reaction dynamics of several molecules in solution by using this method. The photochemistry of iodoform (CHI3) has received considerable attention because of the suggested formation of a unique intermediate, called isoiodoform (CHI2 I), which has been studied by using several time-resolved spectroscopic methods, such as transient absorption and transient resonance Raman spectroscopies. According to these studies, a parent iodoform molecule loses an iodine atom upon photoexcitation at 267 nm. The CHI2 radical and the I atom then recombine geminately to form isoiodoform within the solvent cage; this is a common reaction in diand trihaloalkanes. Previous studies suggest that isoiodoform is the major intermediate, with a lifetime of several microseconds. We note that optical spectroscopy can be highly sensitive to a particular species, but sometimes this advantage translates into biased or nonglobal sampling, that is, certain intermediates might be optically “silent” and escape detection. In contrast, the diffraction signal contains scattering information from all the atoms in the sample, thus providing a global picture of the reactions—although at the expense of sensitivity. Herein we report the global dynamics of the photodissociation of iodoform in methanol and show that the formation of the isomer is not a major reaction channel in the investigated time range, that is, from 100 ps to 3 ms. Time-resolved diffraction data were collected on the pump–probe beamline ID09B at the European Synchrotron Radiation Facility (ESRF) at time delays of 3 ns; 100, 100, and 300 ps; 1, 3, 6, 10, 30, 45, 60, 300, and 600 ns; and 1 and 3 ms. The diffraction signal corresponding the structural change is quite weak (about 0.1%) relative to the total diffraction signal. To extract the structural changes only, a non-excited reference data (at 3 ns) is subtracted from the diffraction data obtained at positive time delays. To magnify the scattered intensities at high angles (at which the signal becomes weak as a result of the decay in the atomic form factors), the change in the diffracted intensity, DS(q), is multiplied by q= (4p/l)sin(q/2), where l is the X-ray wave[*] J. H. Lee, J. Kim, K. H. Kim, Dr. J. Choi, Prof. H. Ihee Center for Time-Resolved Diffraction Department of Chemistry (BK21) Korea Advanced Institute of Science and Technology (KAIST) 335 Gwahangno, Yuseong-gu, Daejeon 305–701 (Republic of Korea) Fax: (+82)42-869-2810 E-mail: hyotcherl.ihee@kaist.ac.kr Homepage: http://time.kaist.ac.kr

Ultrafast structural dynamics of the photocleavage of protein hybrid nanoparticles.

Protein-coated gold nanoparticles in suspension are excited by intense laser pulses to mimic the light-induced effect on biomolecules that occur in photothermal laser therapy with nanoparticles as photosensitizer. Ultrafast X-ray scattering employed to access the nanoscale structural modifications of the protein-nanoparticle hybrid reveals that the protein shell is expelled as a whole without denaturation at a laser fluence that coincides with the bubble formation threshold. In this ultrafast heating mediated by the nanoparticles, time-resolved scattering data show that proteins are not denatured in terms of secondary structure even at much higher temperatures than the static thermal denaturation temperature, probably because time is too short for the proteins to unfold and the temperature stimulus has vanished before this motion sets in. Consequently the laser pulse length has a strong influence on whether the end result is the ligand detachment (for example drug delivery) or biomaterial degradation.

Hole Trapping of G‐Quartets in a G‐Quadruplex

Since reduction and oxidation of DNA are essential processes occurring in various biological phenomena, electron and hole transfer in DNA has drawn recent attention because of its importance and potential application in biological science and nano-biotechnology, respectively. In practice, it is known that the reduction of DNA closely relates to the repair of damaged DNA such as a T-T cyclobutane lesion, whereas the oxidation of DNA promotes oxidative damage, apoptosis, and cancer. Thus, it is important to understand the mechanisms and dynamics of DNA-mediated charge-transfer processes. Excess electron transfer (EET) in DNA has been studied by various techniques, such as laser flash photolysis, g-ray radiolysis, or product analysis which were used to analyze the cleavage of 5-bromo-2’-deoxyuridine (BrdU) or the T-T dimer in DNA by photoinduced electron transfer. The laser flash photolysis is conducted principally on shortlength DNA, which is containing four A-T base pairs between two chromophores. Meanwhile, the DNA-mediated hole transfer occurs over a distance greater than 20 nm and the migration of the hole along the DNA involves many steps of short-distance charge-transfer processes between stacked guanine (G) bases because guanine among the four natural DNA bases is most sensitive to oxidation. The hole transfer rate depends on the inserted nucleobase between the G-C base pairs. In addition, the delocalization of the charge over the stacked G bases along the DNA stem has also been reported. Most studies on DNA-mediated charge-transfer processes have been performed on a DNA duplex with Watson–Crick base pairing. However, recently, non-B DNAs including Gquadruplexes have attracted great attention as fascinating materials for nanotechnology because of their unique structures. Especially, G-quadruplexes formed from various G-rich sequences have received great attention in biological research because in vitro they block the binding of telomerase and act as a transcriptional repressor element and enzyme to provide cancerous cells immortality. Furthermore, G-quadruplexes are an emerging topic for developing DNA-based molecular electronic devices because of their ability as electron carrier, their unique hole-trapping property, and their high conductance. With hole transfer in DNA, recently, hole trapping has also been regarded as an important process to determine the overall efficiency of hole migration in DNA. Here, we have investigated the hole transfer and trapping in a riboflavin-labeled G-quadruplex using femtosecond (fs) laser flash photolysis and pulse radiolysis. In contrast to duplex DNAs, in which p-p stacking has been believed to be a medium for hole transfer, G-quadruplexes have G-quartet structures composed of four G-bases located in the same plane and have also the stacked G-bases (Scheme 1). Thus, it

Reversible Conformational Switching of i‐Motif DNA Studied by Fluorescence Spectroscopy

Non‐B DNAs, which can form unique structures other than double helix of B‐DNA, have attracted considerable attention from scientists in various fields including biology, chemistry and physics etc. Among them, i‐motif DNA, which is formed from cytosine (C)‐rich sequences found in telomeric DNA and the promoter region of oncogenes, has been extensively investigated as a signpost and controller for the oncogene expression at the transcription level and as a promising material in nanotechnology. Fluorescence techniques such as fluorescence resonance energy transfer (FRET) and the fluorescence quenching are important for studying DNA and in particular for the visualization of reversible conformational switching of i‐motif DNA that is triggered by the protonation. Here, we review the latest studies on the conformational dynamics of i‐motif DNA as well as the application of FRET and fluorescence quenching techniques to the visualization of reversible conformational switching of i‐motif DNA in nano‐biotechnology.

100 ps time-resolved solution scattering utilizing a wide-bandwidth X-ray beam from multilayer optics

A new method of time-resolved solution scattering utilizing X-ray multilayer optics is presented.

Radical cation of star-shaped condensed oligofluorenes having isotruxene as a core: importance of rigid planar structure on charge delocalization.

Because of their excellent optical and electronic properties, oligofluorenes and polyfluorenes have been investigated for years. Recently developed star-shaped oligomers bearing a truxene or isotruxene core are interesting two-dimensional oligomers. Since employment of a condensed ring system will be effective in further extension of π-conjugation system, we studied electronic and vibrational properties of radical cation of CITFn, star-shaped condensed oligomer with isotruxene core and fluorene unit, by means of the radiation chemical methods. Absorption spectra of radical cation of CITFn were measured in the wide spectral range, which revealed extended π-conjugation of CITFn. Furthermore, time-resolved resonance Raman spectra during pulse radiolysis revealed that the oxidation of CITFn induced structural change to enhance quinoidal character. The Raman data and theoretical calculation indicated that the rigid framework of the present star-shaped oligomer which makes the oligomer a planar structure is quite important in extension of the conjugation pathway.