Hyun Sun Cho

Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering

We have developed a time-resolved x-ray scattering diffractometer capable of probing structural dynamics of proteins in solution with 100-ps time resolution. This diffractometer, developed on the ID14B BioCARS (Consortium for Advanced Radiation Sources) beamline at the Advanced Photon Source, records x-ray scattering snapshots over a broad range of q spanning 0.02–2.5 Å-1, thereby providing simultaneous coverage of the small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) regions. To demonstrate its capabilities, we have tracked structural changes in myoglobin as it undergoes a photolysis-induced transition from its carbon monoxy form (MbCO) to its deoxy form (Mb). Though the differences between the MbCO and Mb crystal structures are small (rmsd < 0.2 Å), time-resolved x-ray scattering differences recorded over 8 decades of time from 100 ps to 10 ms are rich in structure, illustrating the sensitivity of this technique. A strong, negative-going feature in the SAXS region appears promptly and corresponds to a sudden > 22 Å3 volume expansion of the protein. The ensuing conformational relaxation causes the protein to contract to a volume ∼2 Å3 larger than MbCO within ∼10 ns. On the timescale for CO escape from the primary docking site, another change in the SAXS/WAXS fingerprint appears, demonstrating sensitivity to the location of the dissociated CO. Global analysis of the SAXS/WAXS patterns recovered time-independent scattering fingerprints for four intermediate states of Mb. These SAXS/WAXS fingerprints provide stringent constraints for putative models of conformational states and structural transitions between them.

Photochemistry of covalently-linked multi-porphyrinic systems

Abstract Synthesis, structural characteristics, and optical and electrochemical properties of various covalently-linked porphyrin arrays are described. First, aromatic-spacer bridged diporphyrins were prepared in which the diporphyrin geometries were conformationally-restricted and thus suitable for detailed studies on the exciton coupling and the intramolecular energy and/or electron transfer reactions. Secondly, the Ag(I)-salt oxidation of 5,15-diaryl Zn(II) porphyrins provided meso–meso-linked Zn(II)-diporphyrins. This reaction is advantageous in light of its high regioselectivity and easy extension to longer porphyrin arrays. The doubling reaction was repeated up to the synthesis of a discrete 128-mer, which is, to the best of our knowledge, the longest man-made molecule. Finally, the oxidation of meso–meso-linked Zn(II) porphyrin arrays with a combination of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and Sc(III)(OTf)3 produced fused porphyrin arrays with full π-conjugation, which displayed extremely small HOMO–LUMO gaps that reach into the infrared region.

Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells

Tetrandrine (TET), a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, is known to have anti-tumor activity in various malignant neoplasms. However, the precise mechanism by which TET inhibits tumor cell growth remains to be elucidated. The present studies were performed to characterize the potential effects of TET on phosphoinositide 3-kinase/Akt and extracellular signal-regulated kinase (ERK) pathways since these signaling pathways are known to be responsible for cell growth and survival. TET suppressed cell proliferation and induced apoptosis in A549 human lung carcinoma cells. TET treatment resulted in a down-regulation of Akt and ERK phosphorylation in both time-/concentration-dependent manners. The inhibition of ERK using PD98059 synergistically enhanced the TET-induced apoptosis of A549 cells whereas the inhibition of Akt using LY294002 had a less significant effect. Taken together, our results suggest that TET: i) selectively inhibits the proliferation of lung cancer cells by blocking Akt activation and ii) increases apoptosis by inhibiting ERK. The treatment of lung cancers with TET may enhance the efficacy of chemotherapy and radiotherapy and increase the apoptotic potential of lung cancer cells.

Genotoxicity, acute oral and dermal toxicity, eye and dermal irritation and corrosion and skin sensitisation evaluation of silver nanoparticles

Abstract To clarify the health risks related to silver nanoparticles (Ag-NPs), we evaluated the genotoxicity, acute oral and dermal toxicity, eye irritation, dermal irritation and corrosion and skin sensitisation of commercially manufactured Ag-NPs according to the OECD test guidelines and GLP. The Ag-NPs were not found to induce genotoxicity in a bacterial reverse mutation test and chromosomal aberration test, although some cytotoxicity was observed. In acute oral and dermal toxicity tests using rats, none of the rats showed any abnormal signs or mortality at a dose level of ∼ 2000 mg/kg. Similarly, acute eye and dermal irritation and corrosion tests using rabbits revealed no significant clinical signs or mortality and no acute irritation or corrosion reaction for the eyes and skin. In a skin sensitisation test using guinea pigs, one animal (1/20) showed discrete or patchy erythema, thus Ag-NPs can be classified as a weak skin sensitiser.

Clonidine premedication prevents preoperative hypokalemia.

STUDY OBJECTIVE To test the hypothesis that clonidine premedication could prevent an increase of plasma epinephrine occurring as a result of anxiety, and a decrease of the serum potassium (K+) levels before the induction of anesthesia. DESIGN Randomized, double-blinded study. SETTING University Hospital of Seoul. PATIENTS 44 ASA physical status I and II patients, aged 20 to 50 years, scheduled for knee, ear, or nose surgery. INTERVENTION 44 patients were randomly allocated into one of two groups: 22 patients (clonidine group) received clonidine 300 microg orally at 120 minutes before the induction of anesthesia. The other 22 patients (control group) received a placebo. MEASUREMENTS AND MAIN RESULTS Anxiety level, serum K+, and plasma epinephrine were measured at an outpatient clinic, and immediately before the induction of anesthesia. There were no differences between groups in degree of anxiety experienced, serum K+, or plasma epinephrine levels as measured at the out-patient clinic. Immediately before the induction of anesthesia, the serum K+ levels of the clonidine group were higher than those of the control group (3.89 +/- 0.26 mEq/L vs. 3.50 +/- 0.36 mEq/L), and anxiety and plasma epinephrine levels of clonidine group were lower than those of the control group (p < 0.05). The frequency of hypokalemia (K+ < or = 3.5 mEq/L) of the clonidine group immediately before the induction of anesthesia was significantly lower than that of the control group (0% vs. 50%). CONCLUSIONS Clonidine premedication was effective in preventing hypokalemic episodes occurring before the induction of anesthesia.

Probing anisotropic structure changes in proteins with picosecond time-resolved small-angle X-ray scattering.

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals and suggest that the early time structural dynamics in the crystalline state carry over to proteins in solution.

Physico-chemical characterization-based safety evaluation of nanocalcium

Nano- and microcalcium provided from the KFDA were compared in terms of physico-chemical properties. Calcium samples were tested using EF-TEM and X-ray diffractometry to check for size/morphology and crystal formation, respectively. Two samples of nano- and microcalcium were selected for further evaluation by FE-SEM, DLS (nano-size, 200-500nm; agglomerate, >5 μm; micro-size, 1.5-30 μm), and electron spin resonance. Both samples were heterogeneous in size, existed as single crystal and aggregated form, and did not generate reactive oxygen species. The specific surface area of nano- and microcalcium measured by N2 Brunauere Emmette Teller method was 12.90±0.27 m(2)/g and 1.12±0.19 m(2)/g, respectively. Inductively coupled plasma optical emission spectrometry analysis revealed the release of 2-3 times more calcium ion from nano- compared to microcalcium at pH 5 and 7. Genotoxicity and acute single-dose and repeated-dose 14-day oral toxicity testing in SD rats performed to evaluate the safety of nanocalcium did not reveal toxicity. However, long-term monitoring will be required for an unequivocal conclusion. A nanocalcium dose of 1 g/kg is recommended as the maximum dose for repeated dose 13-week oral toxicity. Further studies could provide details of toxicity of nanocalcium on the repeated dose 13-week oral toxicity test.

Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering

The capacity to respond to environmental changes is crucial to an organisms survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 Å) is significantly larger than that for the ground state (Rg = 14.7 Å) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the β-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.

A microfluidics-based approach for serial time-resolved crystallography

Methods in serial crystallography have shown success for performing time-resolved experiments to visualize macromolecular dynamics in real-time. Such experiments however, require the production of large quantities of isomorphous and uniformly-sized crystals in order to merge diffraction data from numerous crystals and thereby achieve high signal-to-noise structure factor amplitudes. Further, high-throughput data collection is needed to acquire a sufficient amount of data to solve structures over an array of time delays. To meet these needs, we present a microfluidics approach for macromolecular crystallization and room temperature in situ serial data collection that can be utilized at synchrotron or X-ray free electron laser (XFEL) beamlines, and is suited for timeresolved crystallography experiments. Here, ~1000 crystals are grown in a 1 m long glass capillaries inside nanoliter aqueous droplets emulsified in fluorinated oil and stabilized by a surfactant. Droplets of two different sizes (1:5 volume ratio) are generated and are alternatively loaded into the capillary where the smaller droplet contains a 50:50 protein:precipiant mixture and the larger droplet acts as the reservoir containing the mother liquor. Small droplet volumes induce a negative feedback mechanism that causes the growth of one crystal-per-drop with ~35 μm size and uniform characteristics. Crystals are delivered via a syringe pump containing fluorinated oil that acts as a mobile phase into a thin-walled plastic sample cell that has low background scatter for data collection. Utilizing hen egg-white lysozyme (HEWL) we demonstrate the potential of this microfluidics approach for application to a wide range of time-resolved crystallography experiments.

Watching Proteins Function with Time-Resolved X-ray Diffraction

To understand how proteins function, it is crucial to know the time-ordered sequence of structural changes that occur as they execute their designed function. We recently developed on the BioCARS beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in proteins with 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore [1], and geminate ligand-binding dynamics in hemoglobin [2]. Briefly, a picosecond laser pulse photoexcites a protein and triggers a structural change, which is probed with a suitably delayed picosecond X-ray pulse. This “pump-probe” approach recovers time-resolved diffraction “snapshots” whose corresponding electron density maps can be stitched together into a real-time movie of the structural changes that ensue. The mechanistically detailed, near-atomic resolution description of the PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics [3]. This research was supported in part by the Intramural Research Program of the NIH, NIDDK.References:[1] F. Schotte, H.S. Cho, V.R. Kaila, H. Kamikubo, N. Dashdorj, E.R. Henry, T. Graber, R. Henning, M. Wulff, G. Hummer, P.A. Anfinrud Proc. Natl. Acad. Sci. U.S.A. 109, 19256 (2012).[2] F. Schotte, H. S. Cho, J. Soman, M. Wulff, J. S. Olson and P. A. Anfinrud, Chemical Physics, 422, 98-106 (2013).[3] V.R.I. Kaila, F. Schotte, H.S. Cho, G. Hummer, and P.A. Anfinrud, Nature Chemistry, 6, 258 (2014).