Shulin Zhuang

Interactions of benzotriazole UV stabilizers with human serum albumin: Atomic insights revealed by biosensors, spectroscopies and molecular dynamics simulations.

Benzotriazole UV stabilizers (BZTs) belong to one prominent group of ultraviolet (UV) stabilizers and are widely used in various plastics materials. Their large production volumes, frequent detections in the environment and potential toxicities have raised increasing public concern. BZTs can be transported in vivo by transport proteins in plasma and the binding association to transport proteins may serve as a significant parameter to evaluate the bioaccumulative potential. We utilized a novel HSA biosensor, circular dichroism spectroscopy, fluorescence spectroscopy to detect the dynamic interactions of six BZTs (UV-326, UV-327, UV-328, UV-329, UV-P, and BZT) with human serum albumin (HSA), and characterized the corresponding structure-activity relationships (SAR) by molecular dynamics simulations. All test BZTs potently bind at Sudlow site I of HSA with a binding constant of 10(4) L/mol at 298 K. Minor changes in the moieties of BZTs affect their interactions with HSA and differently induce conformations of HSA. Their binding reduced electrochemical impedance spectra and α-helix content of HSA, caused slight red-shifted emission, and changed fluorescence lifetime components of HSA in a concentration-dependent mode. UV-327 and UV-329 form hydrogen bonds with HSA, while UV-329, UV-P and BZT bind HSA with more favorable electrostatic interactions. Our in vitro and in silico study offered a significant framework toward the understanding of risk assessment of BZTs and provides guide for future design of environmental benign BZTs-related materials.

Disruption of the Hormonal Network and the Enantioselectivity of Bifenthrin in Trophoblast: Maternal−Fetal Health Risk of Chiral Pesticides

Endocrine-disrupting chemicals (EDCs) can interfere with normal hormone signaling to increase health risks to the maternal-fetal system, yet few studies have been conducted on the currently used chiral EDCs. This work tested the hypothesis that pyrethroids could enantioselectively interfere with trophoblast cells. Cell viability, hormone secretion, and steroidogenesis gene expression of a widely used pyrethroid, bifenthrin (BF), were evaluated in vitro, and the interactions of BF enantiomers with estrogen receptor (ER) were predicted. At low or noncytotoxic concentrations, both progesterone and human chorionic gonadotropin secretion were induced. The expression levels of progesterone receptor and human leukocyte antigen G genes were significantly stimulated. The key regulators of the hormonal cascade, GnRH type-I and its receptor, were both upregulated. The expression levels of selected steroidogenic genes were also significantly altered. Moreover, a consistent enantioselective interference of hormone signaling was observed, and S-BF had greater effects than R-BF. Using molecular docking, the enantioselective endocrine disruption of BF was predicted to be partially due to enantiospecific ER binding affinity. Thus, BF could act through ER to enantioselectively disturb the hormonal network in trophoblast cells. These converging results suggest that the currently used chiral pesticides are of significant concern with respect to maternal-fetal health.

Molecular interactions of benzophenone UV filters with human serum albumin revealed by spectroscopic techniques and molecular modeling

Benzophenone (BP)-type UV filters have been widely used in many personal care products to protect human from UV exposure. Their dermal applications can cause direct human health risk following accumulation in bloodstream. Few studies have addressed whether BP-type UV filters could bind and alter the structure and function of human serum albumin (HSA), the major carrier protein in plasma. Four benzophenones, BP-1, BP-2, BP-3 and BP-8 were selected to investigate their potentially toxic interactions with HSA and the intrinsic binding mechanism using combined spectroscopies and molecular docking techniques. Four benzophenones significantly quench the intrinsic fluorescence of HSA via static mode. The competitive binding fluorescence assay and molecular docking both revealed that the benzophenones bind at site II of HSA. Their binding constants range from 1.91 × 10(4)M(-1) to 12.96 × 10(4)M(-1) at 296 K. BP-8 interacts with HSA mainly through hydrogen bonding interactions and van der Waals interactions, while hydrophobic interactions and electrostatic interactions are dominant for interactions between BP-1, BP-2, BP-3 and HSA. Molecular docking revealed that the changes in structural moiety and hydrophobicity of four benzophenones account for their different binding affinities. As further revealed by circular dichroism and time-resolved fluorescence decay, these benzophenones cause global and local structural changes of HSA, which illustrates their potential toxicity to cause structural damage of HSA. Two degradation products of BP-3 have higher binding affinities to HSA, suggesting higher potencies in causing adverse effects on human health.

Probing the molecular interaction of triazole fungicides with human serum albumin by multispectroscopic techniques and molecular modeling.

Triazole fungicides, one category of broad-spectrum fungicides, are widely applied in agriculture and medicine. The extensive use leads to many residues and casts potential detrimental effects on aquatic ecosystems and human health. After exposure of the human body, triazole fungicides may penetrate into the bloodstream and interact with plasma proteins. Whether they could have an impact on the structure and function of proteins is still poorly understood. By using multispectroscopic techniques and molecular modeling, the interaction of several typical triazole fungicides with human serum albumin (HSA), the major plasma protein, was investigated. The steady-state and time-resolved fluorescence spectra manifested that static type, due to complex formation, was the dominant mechanism for fluorescence quenching. Structurally related binding modes speculated by thermodynamic parameters agreed with the prediction of molecular modeling. For triadimefon, hydrogen bonding with Arg-218 and Arg-222 played an important role, whereas for imazalil, myclobutanil, and penconazole, the binding process was mainly contributed by hydrophobic and electrostatic interactions. Via alterations in three-dimensional fluorescence and circular dichroism spectral properties, it was concluded that triazoles could induce slight conformational and some microenvironmental changes of HSA. It is anticipated that these data can provide some information for possible toxicity risk of triazole fungicides to human health and be helpful in reinforcing the supervision of food safety.

Impedance sensing and molecular modeling of an olfactory biosensor based on chemosensory proteins of honeybee.

By mimicking biological olfaction, biosensors have been used for the detection of important ligands in complex environments. An olfactory biosensor based on chemosensory proteins (CSPs) was designed by immobilizing honeybee CSPs (Ac-ASP3) on the interdigitated golden electrodes. Its responses to ligands of pheromones and floral odors were recorded by impedance spectroscopy. The relative decrease of charge transfer resistance of the biosensor is proportional to the logarithm of ligand concentration from 10(-7)M to 10(-3)M. To explore the molecular recognition processes of the biosensor, the tertiary structure of the protein was modeled and the protein-ligand interactions were investigated by the molecular docking. Our docking results verified the validity of experiments and showed that the specific ligands could form hydrogen bonds with some of the conserved residues, such as Cys 60 and Gln 64 of Ac-ASP3. Furthermore, combining the molecular modeling with impedance detection, the accuracy, specificity and predictability of the ligands binding to the protein could be improved. Thus, CSPs will provide a promising approach for chemical molecular sensing at low concentrations.

Atomic Insights into Distinct Hormonal Activities of Bisphenol A Analogues toward PPARγ and ERα Receptors

Bisphenol A analogues (BPAs) belong to a wide variety of large volume chemicals with diverse applications yet emerging environmental concerns. Limited experimental data have demonstrated that BPAs with different halogenation patterns distinctly affect the agonistic activities toward proliferator-activated receptor (PPAR)γ and estrogen receptors (ER)α. Understanding the modes of action of BPAs toward different receptors is essential, however, the underlying molecular mechanism is still poorly understood. Here we probed the molecular recognition process of halogenated BPAs including TBBPA, TCBPA, BPAF, BPC, triBBPA, diBBPA, and monoBBPA toward PPARγ and ERα by molecular modeling, especially the impact of different halogen patterns. Increasing bromination at phenolic rings of BPAs was found highly correlated with electrostatic interactions (R(2) = 0.978 and 0.865 toward PPARγ and ERα, respectively) and van der Waals interactions (R(2) = 0.995 and 0.994 toward PPARγ and ERα, respectively). More halogenated phenolic rings at 3,5-positions of BPAs increase the shielding of the hormonally active phenolic OH and markedly decrease electrostatic interactions favorable for agonistic activities toward PPARγ, but unfavorable for agonistic activities toward ERα. The halogenation at the phenolic rings of BPAs exerts more impact on molecular electrostatic potential distribution than halogenation at the bridging alkyl moiety. Different halogenations further alter hydrogen bond interactions of BPAs and induce conformational changes of PPARγ ligand binding domain (LBD) and ERα LBD, specifically affecting the stabilization of helix H12 attributable to the different agonistic activities. Our results indicate that structural variations in halogenation patterns result in different interactions of BPAs with PPARγ LBD and ERα LBD, potentially causing distinct agonistic/antagonistic toxic effects. The various halogenation patterns should be fully considered for the design of future environmentally benign chemicals with reduced toxicities and desired properties.

Enantioselective endocrine-disrupting effects of bifenthrin on hormone synthesis in rat ovarian cells.

Bifenthrin (BF), a broad-spectrum and widely used synthetic pyrethroid, is a typical chiral pesticide. More attention is being paid to the health risk assessment of the enantioselective toxicity of BF isomers. In this study, we used rat ovarian granulosa cells as in vitro model to investigate effects of BF enantiomers on the biosynthesis of two hormones, progesterone and prostaglandin E2 (PGE2), which are critical for mammalian reproduction. We showed that 1S-cis-BF, but not 1R-cis-BF significantly decreased the secretion of progesterone and PGE2 in granulosa cells. 1S-isomer of BF reduced the expression of genes P450scc, StAR, PBR and DBI, as well as COX-2, which are involved in regulating the rate-limiting steps of progesterone or PGE2 biosynthesis. The transcriptional activation of StAR and COX-2 promoter were disrupted by 1S-cis-BF. Furthermore, activity of protein kinase C (PKC), an important signaling mediator of progesterone and PGE2 synthesis, was differentially inhibited by 1S-cis-BF. The data of molecular docking revealed that one hydrogen bond was formed between 1S-cis-BF and PKC protein. In conclusion, we firstly reported in this study the enantioselective disrupting effects of BF isomers on progesterone and PGE2 synthesis via PKC pathway in rat ovarian cells. Our findings suggest that the enantioselective toxicity of chiral pesticides should be considered for evaluating mammalian reproductive health, a toxicologic endpoint of great concern in health risk assessment.

Protein-Protein Interaction Regulates Proteins' Mechanical Stability

Elastomeric proteins are molecular springs found not only in a variety of biological machines and tissues, but also in biomaterials of superb mechanical properties. Regulating the mechanical stability of elastomeric proteins is not only important for a range of biological processes, but also critical for the use of engineered elastomeric proteins as building blocks to construct nanomechanical devices and novel materials of well-defined mechanical properties. Here we demonstrate that protein-protein interactions can potentially serve as an effective means to regulate the mechanical properties of elastomeric proteins. We show that the binding of fragments of IgG antibody to a small protein, GB1, can significantly enhance the mechanical stability of GB1. The regulation of the mechanical stability of GB1 by IgG fragments is not through direct modification of the interactions in the mechanically key region of GB1; instead, it is accomplished via the long-range coupling between the IgG binding site and the mechanically key region of GB1. Although Fc and Fab bind GB1 at different regions of GB1, their binding to GB1 can increase the mechanical stability of GB1 significantly. Using alanine point mutants of GB1, we show that the amplitude of mechanical stability enhancement of GB1 by Fc does not correlate with the binding affinity, suggesting that binding affinity only affects the population of GB1/human Fc (hFc) complex at a given concentration of hFc, but does not affect the intrinsic mechanical stability of the GB1/hFc complex. Furthermore, our results indicate that the mechanical stability enhancement by IgG fragments is robust and can tolerate sequence/structural perturbation to GB1. Our results demonstrate that the protein-protein interaction is an efficient approach to regulate the mechanical stability of GB1-like proteins and we anticipate that this new methodology will help to develop novel elastomeric proteins with tunable mechanical stability and compliance.

Insights into unbinding mechanisms upon two mutations investigated by molecular dynamics study of GSK3β–axin complex: Role of packing hydrophobic residues

Glycogen synthase kinase 3β (GSK 3β) is a key component of several cellular processes including Wnt and insulin signalling pathways. The interaction of GSK3β with scaffolding peptide axin is thought to be responsible for the effective phosphorylation of β‐catenin, the core effector of Wnt signaling, which has been linked with the occurrence of colon cancer and melanoma. It has been demonstrated that the binding of axin to GSK3β is abolished by the single‐point mutation of Val267 to Gly (V267G) in GSK3β or Leu392 to Pro (L392P) in axin. Molecular dynamics (MD) simulations were performed on wild type (WT), V267G mutant and L392P one to elucidate the two unbinding mechanisms that occur through different pathways. Besides, rough energy and residue‐based energy decomposition were calculated by MM_GBSA (molecular mechanical Generalized_Born surface area) approach to illuminate the instability of the two mutants. The MD simulations of the two mutants and WT reveal that the structure of GSK3β remains unchanged, while axin moves away from the interfacial hydrophobic pockets in both two mutants. Axin exhibits positional shift in V267G mutant, whereas, losing the hydrogen bonds that are indispensable for stabilizing the helix structure of wild type axin, the helix of axin is distorted in L392P mutant. To conclude, both two mutants destroy the hydrophobic interaction that is essential to the stability of GSK3β‐axin complex. Proteins 2007.

Olfactory biosensor for insect semiochemicals analysis by impedance sensing of odorant-binding proteins on interdigitated electrodes.

Insects can sensitively and selectively detect thousands of semiochemicals at very low concentrations by their remarkable olfactory systems. As one of the most important olfactory proteins, odorant-binding proteins (OBPs) from insects are the most promising candidates for fabricating biosensors to detect biochemical molecules in the chemical ecology as well as for other biotechnological applications. In this study, we designed an olfactory biosensor by immobilizing OBPs from oriental fruit fly on interdigitated electrodes to detect semiochemicals. After successfully separated and purified, OBPs were immobilized by the special designed polyethylene glycol (PEG), SH-PEG-COOH, to produce a robust sensing membrane. Based on electrochemical sensing, interactions between OBPs and different semiochemicals emitted from host plants of the insect, such as the isoamyl acetate, β-ionone, and benzaldehyde, could be sensitively detected. With related amino acid residues in the hydrophobic cavities distinguished, the interaction forces between semiochemicals and OBPs were analyzed by molecular docking. Integrated biological olfaction proteins of insects, OBPs based biosensors could not only advance the progress in the understanding of chemical communication systems of insects, but also show promising potentials for biosensing applications in many fields.