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Ramachandran P.L.,University of Oxford | Lovett J.E.,University of Oxford | Lovett J.E.,University of Edinburgh | Carl P.J.,Bruker Biospin Gmbh | And 7 more authors.
Journal of the American Chemical Society | Year: 2011

The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Å upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from 1H-15N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I2′ state (also referred to as the pB state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I 2′ state, which is structurally modeled by dynamical annealing and refinement calculations. © 2011 American Chemical Society.

Lee J.S.,KAIST | Yoon I.,KAIST | Kim J.,Center for Time Resolved Diffraction | Ihee H.,Center for Time Resolved Diffraction | And 2 more authors.
Angewandte Chemie - International Edition | Year: 2011

Down to the wire: A simple vapor-transport process using linear diphenylalanine as the starting material has resulted in the self-assembly of cyclodipeptide nanowires with an orthorhombic symmetry. Furthermore, the single-crystalline nanowires exhibit a strong blue luminescence centered at 465 nm and possess semiconducting properties (see picture). © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kim K.H.,Center for Time Resolved Diffraction | Ok T.,Institute Pasteur Korea | Ok T.,KAIST | Lee K.,Korea University | And 4 more authors.
Journal of the American Chemical Society | Year: 2010

Herein we present the long-sought quantitative catalyst-substrate association relationships based on experimentally measured quantitative association preferences of diverse metathesis Mo and Ru catalysts (Mo-1, Schrock Mo; Mo-2, Schrock-Hoveyda Mo; Ru-1, Grubbs first generation Ru; Ru-2, Grubbs second generation Ru; Ru-3:, Grubbs-Hoveyda first generation Ru; and Ru-4, Grubbs-Hoveyda second generation Ru) to their substrates (alkenes, alkynes and allenes), determined directly by a general method based on FRET principle. The determined substrate preferences are proved to be dependent on the molecular identity of the catalyst, exhibiting the preference order of alkyne > alkene > allene for Mo-1 and Mo-2, allene > alkene > alkyne for Ru-1 and Ru-3, and alkyne > allene > alkene for Ru-2 and Ru-4. The results enable us to probe metathesis mechanisms by answering issues in metathesis reactions including the controversial reaction initiation in enyne or allenyne metathesis. © 2010 American Chemical Society.

Kim T.W.,Center for Time Resolved Diffraction | Lee J.H.,Center for Time Resolved Diffraction | Choi J.,Center for Time Resolved Diffraction | Kim K.H.,Center for Time Resolved Diffraction | And 10 more authors.
Journal of the American Chemical Society | Year: 2012

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. © 2012 American Chemical Society.

Ibrahimkutty S.,Karlsruhe Institute of Technology | Kim J.,Center for Time Resolved Diffraction | Cammarata M.,European Synchrotron Radiation Facility | Ewald F.,European Synchrotron Radiation Facility | And 4 more authors.
ACS Nano | Year: 2011

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. © 2011 American Chemical Society.

Kim K.H.,Center for Time Resolved Diffraction | Muniyappan S.,Center for Time Resolved Diffraction | Oang K.Y.,Center for Time Resolved Diffraction | Kim J.G.,Center for Time Resolved Diffraction | And 12 more authors.
Journal of the American Chemical Society | Year: 2012

Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I 1, I 2, and I 3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme-heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I 1 intermediate is generated within 100 ps and transforms to the R-like I 2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I 3 intermediate is formed via subunit rotation, a decrease in the heme-heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I 3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme-heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics. © 2012 American Chemical Society.

Choi J.,Center for Time Resolved Diffraction | Yang C.,Center for Time Resolved Diffraction | Kim J.,Center for Time Resolved Diffraction | Ihee H.,Center for Time Resolved Diffraction
Journal of Physical Chemistry B | Year: 2011

We investigate optically triggered protein folding dynamics of cytochrome c (Cytc) using transient grating (TG) and transient absorption (TA) spectroscopies. Despite many studies on protein folding dynamics of Cytc, a well-known model protein, direct spectroscopic evidence for the three-dimensional global folding process has been rarely reported. By measuring the TG signal of CO-bound Cytc (Cytc-CO) in the presence of a denaturant, we clearly detected the change of diffusion coefficient that reflects the size change of Cytc upon photodissociation of the CO ligand from unfolded Cytc-CO. The quantitative analysis of TG signals supports that the optically triggered folding reaction of Cytc in the presence of a denaturant takes place through a detectable intermediate (three-state folding kinetics). This is in contrast with the two-state folding dynamics of Cytc under a denaturant-free environment without any detectable intermediate.(1)From the quantitative global analysis of the TG signals, the rate constants for the U → I and I → N transitions in a CAPS buffer solution (pH 7) at room temperature in the presence of a denaturant at various concentrations are determined to be 1065 ± 17 to 3476 ± 103 s-1 and 101 ± 6 to 589 ± 21 s -1, respectively. In addition, the activation energies (E a) for the U → I and I → N transitions are determined to be 8.7 ± 1.0 kcal/mol and 7.1 ± 1.3 kcal/mol, respectively. The folding dynamics of Cytc initiated by the CO photolysis is discussed based in terms of the protein size change. © 2011 American Chemical Society.

Kim J.,Center for Time Resolved Diffraction | Kim K.H.,Center for Time Resolved Diffraction | Kim J.G.,Center for Time Resolved Diffraction | Kim T.W.,Center for Time Resolved Diffraction | And 2 more authors.
Journal of Physical Chemistry Letters | Year: 2011

Anisotropic X-ray scattering patterns of transiently aligned protein molecules in solution are measured by using pump-probe X-ray solution scattering. When a linearly polarized laser pulse interacts with an ensemble of molecules, the population of excited molecules is created with their transition dipoles preferentially aligned along the laser polarization direction. We measured the X-ray scattering from the myoglobin protein molecules excited by a linearly polarized, short laser pulse and obtained anisotropic scattering patterns on a 100 ps time scale. An anisotropic scattering pattern contains higher structural information content than a typical isotropic pattern available from randomly oriented molecules. In addition, multiple independent diffraction patterns measured by using various laser polarization orientations will give a substantially increased amount of structural information compared with that from a single isotropic pattern. By monitoring the temporal change of the anisotropic scattering pattern from 100 ps to 1 μs, we observed the orientational dynamics of photogenerated myoglobin with the rotational diffusion time of ∼15 ns. © 2011 American Chemical Society.

Kim K.H.,Center for Time Resolved Diffraction | Oang K.Y.,Center for Time Resolved Diffraction | Kim J.,Center for Time Resolved Diffraction | Lee J.H.,Center for Time Resolved Diffraction | And 2 more authors.
Chemical Communications | Year: 2011

Here we report structural dynamics of equine myoglobin (Mb) in response to the CO photodissociation visualized by picosecond time-resolved X-ray solution scattering. The data clearly reveal new structural dynamics that occur in the timescale of ∼360 picoseconds (ps) and ∼9 nanoseconds (ns), which have not been clearly detected in previous studies. © 2011 The Royal Society of Chemistry.

Kim J.,Center for Time Resolved Diffraction | Kim T.K.,Pusan National University | Ihee H.,Center for Time Resolved Diffraction
Journal of Physical Chemistry A | Year: 2011

Quantum chemical calculations of CF3Br and the CF3 radical are performed using density functional theory (DFT) and time-dependent DFT (TDDFT). Molecular structures, vibrational frequencies, dipole moment, bond dissociation energy, and vertical excitation energies of CF3Br are calculated and compared with available experimental results. The performance of six hybrid and five hybrid meta functionals in DFT and TDDFT calculations are evaluated. The ωB97X, B3PW91, and M05-2X functionals give very good results for molecular structures, vibrational frequencies, and vertical excitation energies, respectively. The ωB97X functional calculates well the dipole moment of CF3Br. B3LYP, one of the most widely used functionals, does not perform well for calculations of the C - Br bond length, bond dissociation energy, and vertical excitation energies. Potential energy curves of the low-lying excited states of CF3Br are obtained using the multiconfigurational spin-orbit ab initio method. The crossing point between 2A1 and 3E states is located near the C - Br bond length of 2.45 Å. Comparison with CH3Br shows that fluorination does not alter the location of the crossing point. The relation between the calculated potential energy curves and recent experimental result is briefly discussed. © 2011 American Chemical Society.

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