Browsing by Author "Tzou, Der-Lii M."

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Magnesium and calcium reveal different chelating effects in a steroid compound: A model study of prednisolone using NMR spectroscopy
(2019-10-01)
Carillo, Kathleen D.
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Wu, Danni
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Lin, Su-Ching
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Tsai, Shen-Long
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Shie, Jiun-Jie
In this work, we used high resolution NMR spectroscopy to investigate metal cation chelation by the steroidal drug 1,4-pregnadiene-11 beta,17 alpha,21-triol-3,20-dione (Prednisolone; abbreviated as Prd). Prd/MgCl2 and Prd/CaCl2 mixtures were prepared at eight different molar ratios. Using two-dimensional H-1/C-13 heteronuclear correlation spectroscopy, we were able to resolve most of the H-1 signals, except those at 1.4-1.55 ppm, where signals for H15 beta, H16 alpha and Me-19 are superimposed. The chelation sites were determined by the cation concentration-dependent C-13 signals. Both ring A and ring D of Prd were found to be involved in Mg2+ chelation, whereas only ring A was involved in Ca2+ chelation. The dihedral angles deduced from the (3)J(H)(-H) coupling constants indicated that ring D of Prd might undergo rather small, but different, distortions in the presence of Mg2+ and Ca2+. Additionally, using the continuous variation method, we deduced that the stoichiometric ratios of the Prd/Mg2+ and Prd/Ca2+ complexes were 1:1 and 2:1, respectively. All of the evidence led us to conclude that the Prd/Mg2+ and Prd/Ca2+ complexes are mediated by different chelating mechanisms.
Solid-state nuclear magnetic resonance investigation of neurosteroid compounds and magnesium interactions
(2019-10-01)
Wu, Danni
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Carillo, Kathleen Joyce D.
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Tsai, Shen-Long
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Shie, Jiun-Jie
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Tzou, Der-Lii M.
The neurosteroid trans-dehydroandrosterone (DHEA) and its analogs with slightly different modifications in the side chain attached to C17, that is, (3S)-acetoxypregn-5-en-20-one (1) and (3S,20R)-acetoxypregn-5-en-20-ol (2), have been synthesized to investigate DHEA-cation interactions. In this study, we applied solid-state H-1/C-13 cross-polarization/magic-angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy to a series of DHEA analog/Mg2+ mixtures at different Mg2+ concentrations. The high-resolution C-13 NMR spectra of 1/Mg2+ mixtures exhibit two distinct C-13 spectral patterns, one attributable to 1 free from Mg2+, and the other attributable to 1 with bound Mg2+. For 2, the C-13 NMR spectra exhibit three distinct spectral patterns; besides that of the free form, the other two can be assigned to Mg2+-bound forms. Based on the analysis of the chemical shift deviations (CSDs), we conclude that both 1 and 2 might be subject to a cation-pi interaction via the C5-C6 double bond, in contrast to that observed previously for DHEA. As demonstrated, DHEA possesses two Mg2+ binding sites, that is, C17-O and C5-C6 double bond, in which the binding affinity of the former is at least three times stronger than that of the latter. The solid-state C-13 NMR investigation allows better understanding of the underlying cation binding effects of neurosteroid molecules in vitro.
The Effect of Calcium and Halide Ions on the Gramicidin A Molecular State and Antimicrobial Activity
(2020-09-01)
Carillo, Kathleen D.
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Lo, Chi-Jen
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Tzou, Der-Lii M.
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Lin, Yi-Hung
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Fang, Shang-Ting
Gramicidin A (gA) forms several convertible conformations in different environments. In this study, we investigated the effect of calcium halides on the molecular state and antimicrobial activity of gramicidin A. The molecular state of gramicidin A is highly affected by the concentration of calcium salt and the type of halide anion. Gramicidin A can exist in two states that can be characterized by circular dichroism (CD), mass, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. In State 1, the main molecular state of gramicidin A is as a dimer, and the addition of calcium salt can convert a mixture of four species into a single species, which is possibly a left-handed parallel double helix. In State 2, the addition of calcium halides drives gramicidin A dissociation and denaturation from a structured dimer into a rapid equilibrium of structured/unstructured monomer. We found that the abilities of dissociation and denaturation were highly dependent on the type of halide anion. The dissociation ability of calcium halides may play a vital role in the antimicrobial activity, as the structured monomeric form had the highest antimicrobial activity. Herein, our study demonstrated that the molecular state was correlated with the antimicrobial activity.
Vaccinia viral A26 protein is a fusion suppressor of mature virus and triggers membrane fusion through conformational change at low pH
(2019-06-01)
Chang, Hung-Wei
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Yang, Cheng-Han
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Luo, Yu-Chun
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Su, Bo-Gang
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Cheng, Huei-Yin
Vaccinia mature virus requires A26 envelope protein to mediate acid-dependent endocytosis into HeLa cells in which we hypothesized that A26 protein functions as an acid-sensitive membrane fusion suppressor. Here, we provide evidence showing that N-terminal domain (aa1-75) of A26 protein is an acid-sensitive region that regulates membrane fusion. Crystal structure of A26 protein revealed that His48 and His53 are in close contact with Lys47, Arg57, His314 and Arg312, suggesting that at low pH these His-cation pairs could initiate conformational changes through protonation of His48 and His53 and subsequent electrostatic repulsion. All the A26 mutant mature viruses that interrupted His-cation pair interactions of His48 and His 53 indeed have lost virion infectivity. Isolation of revertant viruses revealed that second site mutations caused frame shifts and premature termination of A26 protein such that reverent viruses regained cell entry through plasma membrane fusion. Together, we conclude that viral A26 protein functions as an acid-sensitive fusion suppressor during vaccinia mature virus endocytosis. Author summary Vaccinia virus is a complex large DNA virus with a large number of viral membrane proteins to facilitate cell entry. Although it is well established that vaccinia mature virus uses endocytosis to enter cells, it remains unclear how it triggers membrane fusion in the acidic environment of endosomes. Recently, we hypothesized that A26 protein in vaccinia mature virus functions as an acid-sensitive membrane fusion suppressor, which suggests a novel viral regulation not present in other enveloped viruses. We postulated that conformational changes of A26 protein at low pH result in de-repression of viral fusion complex activity to trigger viral and endosomal membrane fusion. Here, we provide structural, biochemical and biological evidence demonstrating that vaccinia A26 protein does indeed function as an acid-sensitive fusion suppressor protein to regulate vaccinia mature virus membrane fusion during endocytosis. Our data reveal an important and unique checkpoint for vaccinia mature virus endocytosis that has not been described for other viruses. Furthermore, by isolating adaptive vaccinia mutants that escaped endocytic blockage, we discovered that mutations within the A26L gene serve as an effective strategy for switching the viral infection route from endocytosis to plasma membrane fusion, expanding viral host range.