Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques

This work was intended to develop self-assembly lipids for incorporating G-protein coupled receptors (GPCRs) in order to improve the success rate for nuclear magnetic resonance spectroscopy (NMR) structural elucidation. We hereby report the expression and purification of uniformly 15N-labeled human cannabinoid receptor-2 domain in insect cell media. The domain was refolded by screening several membrane mimetic environments. Different q ratios of isotropic bicelles were screened for solubilizing transmembrane helix 6, 7 and 8 (TMH67H8). As the concentration of dimyristoylphosphocholine (DMPC) was increased such that the q ratio was between 0.16 and 0.42, there was less crowding in the cross peaks with increasing q ratio. In bicelles of q = 0.42, the maximum number of cross peaks were obtained and the cross peaks were uniformly dispersed. The receptor domain in bicelles beyond q = 0.42 resulted in peak crowding. These studies demonstrate that GPCRs folding especially in bicelles is protein-specific and requires the right mix of the longer chain and shorter chain lipids to provide the right environment for proper folding. These findings will allow further development of novel membrane mimetics to provide greater diversity of lipid mixtures than those currently being employed for GPCR stability and folding, which are critical for both X-ray and NMR studies of GPCRs

Targeting the Endocannabinoid System for Neuroprotection: A 19F-NMR Study of a Selective FAAH Inhibitor Binding with an Anandamide Carrier Protein, HSA

Fatty acid amide hydrolase (FAAH), the enzyme involved in the inactivation of the endocannabinoid anandamide (AEA), is being considered as a therapeutic target for analgesia and neuroprotection. We have developed a brain permeable FAAH inhibitor, AM5206, which has served as a valuable pharmacological tool to explore neuroprotective effects of this class of compounds. In the present work, we characterized the interactions of AM5206 with a representative AEA carrier protein, human serum albumin (HSA), using 19F nuclear magnetic resonance (NMR) spectroscopy. Our data showed that as a drug carrier, albumin can significantly enhance the solubility of AM5206 in aqueous environment. Through a series of titration and competitive binding experiments, we also identified that AM5206 primarily binds to two distinct sites within HSA. Our results may provide insight into the mechanism of HSA-AM5206 interactions. The findings should also help in the development of suitable formulations of the lipophilic AM5206 and its congeners for their effective delivery to specific target sites in the brain

In-situ P-31 MAS NMR probing of the active centers in Ti silicalite molecular sieve

The investigation of the active centers in titanium zeolite was conducted by a probe reaction, the oxidation of trimethylphosphine. The different sites of TS-1 zeolite can be clearly distinguished through P-31 solid-state MAS NMR spectra of adsorbed trimethylphosphine. The results showed that only the trimethylphosphine bound to the coordinate-unsaturated framework Ti sites and the distorted tetrahedral Ti sites ofTS-1 zeolite could be easily oxidized by urea hydrogen peroxide. Diffuse Reflectance UV-Vis and Si-29 MAS NMR spectra further confirmed that the titanium peroxide species (TiOOH), which is formed through the solid-solid interaction between urea-H2O2 and TS-1 zeolite, could be catalytic active centers during the oxidation of trimethylphosphine. (C) 2015 Elsevier B.V. All rights reserved

Oxyresveratrol from mulberry branch extract protects HUVECs against oxidized Low-density Lipoprotein-induced oxidative injury via activation of the Nrf-2/HO-1 pathway

Mulberry branch (Morus spp.) is an agricultural byproduct enriched with polyphenol and flavone. Mulberry branch extract has been reported to protect human umbilical vein endothelial cells (HUVECs) from oxidized low-density lipoprotein (ox-LDL) injury. However, the active ingredient derived from mulberry branch extract responsible for the protective action and the specific mechanism remain to be clarified. In the current study, the main active ingredient with anti-oxidant activity of mulberry branch extract was rapid-screened using the DPPH-HPLC method. Subsequently, the chemical structure was solved with mass spectrometry and nuclear magnetic resonance spectroscopy. Cell viability, levels of malondialdehyde (MDA) and superoxide dismutases (SODs) were measured to evaluate the protective action of the ingredient for HUVECs against ox-ldl-induced injury. DHE and DCFH-DA fluorescent probes were used to measure the level of intracellular reactive oxygen species (ROS). Real-time qPCR and Western blot were conducted to detect mRNA and protein levels. We found that the Oxyresveratrol was the main anti-oxidant ingredient that protecetd HUVECs against ox-ldl-induced injury. The protective action might be achieved via the up-regulation of the Nrf-2/HO-1 signaling and reduction of ROS production. Futhermore, the protective effect of Oxyresveratrol could be reversed by suppression of Nrf-2. Molecular docking analysis revealed strong binding between Oxyresveratrol and Keap-1/Nrf-2 protein complex, mainly via hydrogen bonds linking the specific amino acid residues on the target proteins. Molecular-dynamics simulation suggested that Oxyresveratrol led to dissociation of Nrf-2 from Keap-1 via binding to the Keap-1/Nrf-2 complex. In all, the current study showed that Oxyresveratrol, an active ingredient of mulberry branch extract, could protect HUVECs against the ox-ldl-induced oxidative injury via the dissociation of Nrf-2 from the Keap-1/Nrf-2 to axtivate the Nrf-2/HO-1 signaling pathway. Thus, the Oxyresveratrol might be potent in prevention and treatment of atherosclerosis

Face-Centered-Cubic Large-Pore Periodic Mesoporous Organosilicas with Unsaturated and Aromatic Bridging Groups

Large-pore ethenylene-bridged (−CHCH−) and phenylene-bridged (−C₆H₄−) periodic mesoporous organosilicas (PMOs) with face-centered-cubic structure (<i>Fm</i>3<i>m</i> symmetry) of spherical mesopores were synthesized at 7 °C at low acid concentration (0.1 M HCl) using Pluronic F127 triblock copolymer surfactant in the presence of aromatic swelling agents (1,3,5-trimethylbenzene, xylenes–isomer mixture, and toluene). In particular, this work reports an unprecedented block-copolymer-templated well-ordered ethenylene-bridged PMO with cubic structure of spherical mesopores and an unprecedented block-copolymer-templated face-centered cubic phenylene-bridged PMO, which also has an exceptionally large unit-cell size and pore diameter. The unit-cell parameters of 30 and 25 nm and the mesopore diameters of 14 and 11 nm (nominal BJH-KJS pore diameters of 12–13 and 9 nm) were obtained for ethenylene-bridged and phenylene-bridged PMOs, respectively. Under the considered reaction conditions, the unit-cell parameters and pore diameters were found to be similar when the three different methyl-substituted benzene swelling agents were employed, although the degree of structural ordering appeared to improve for phenylene-bridged PMOs in the sequence of decreased number of methyl groups on the benzene ring