Binsheng Yang

Investigation on the Inclusion Behavior of ApoCopC with Vitamin B6

In neutral phosphate buffer solution of pH 7.0, the interaction between apoCopC and Vitamin B6 has been investigated in detail by means of fluorescence spectroscopy. According to the change of Vitamin B6 fluorescence spectra and fluorescence polarization, we can conclude that a novel supramolecular system is generated. ApoCopC can form a 1:5 host-guest inclusion supramolecular complex with Vitamin B6, and the formation constant has been calculated to be (2.24 ± 0.08) × 104 M− 1. It suggests the strong inclusion ability of apoCopC to the guest molecules. In addition, the mechanism of the apoCopC protein fluorescence quenching by Vitamin B6 was also discussed. And based on the Stern-Volmer equation, the apparent quenching constant was estimated to be (2.75 ± 0.05) × 104 M− 1.

Characterization of self-assembly of Euplotes octocarinatus centrin

Centrin, an EF-hand calcium-binding protein with high homology to calmodulin (CaM), is an essential component for microtubule-organizing center (MTOC) in organisms ranging from algae and yeast to human. It plays an important structural role by contributing to the formation of Ca(2+)-sensitive contractile filaments and some super-molecular assemblies. Previous work suggests that the N-terminal domain of centrin especially its first 20-residue fragment, is required for the self-assembly of protein. Native polyacrylamide gel electrophoresis (native-PAGE), pull-down assay, fluorescence resonance light scattering (RLS) and yeast two-hybrid assay indicate that the C-terminal domain of Euplotes octocarinatus centrin (EoCen) also contributes to the centrin self-assembly besides its N-terminal domain in vivo and in vitro. On the basis of our results, a self-assembly mode of centrin, which is C-to-C as well as N-to-N (between C- and C-terminal domains as well as between N- and N-terminal domains), is put forward providing maybe some insights into the molecular mechanism of centrin functions in the cell.

Naphthol-based fluorescent sensors for aluminium ion and application to bioimaging

Three naphthol Schiff base-type fluorescent sensors, 1,3-Bis(2-hydroxy-1-naphthylideneamino)propane (L1), 1,3-Bis(1-naphthylideneamino)-2-hydroxypropane (L2) and 1,3-Bis(2-hydroxy-1-naphthylideneamino)-2-hydroxypropane (L3), have been synthesized. Their recognition abilities for Al(3+) are studied by fluorescence spectra. Coordination with Al(3+) inhibited the CN isomerization of Schiff base which intensely increase the fluorescence of L1-L3. Possessing a suitable space coordination structure, L3 is a best selective probe for Al(3+) over other metal ions in MeOH-HEPES buffer (3/7, V/V, pH=6.6, 25°C, λem=435nm). A turn-on ratio over 140-fold is triggered with the addition of 1.0 equiv. Al(3+) to L3. The binding constant Ka of L3-Al(3+) is found to be 1.01×10(6.5)M(-1) in a 1:1 complex mode. The detection limit for Al(3+) is 0.05μM. Theoretical calculations have also been included in support of the configuration of the L3-Al(3+) complex. Importantly, the probe L3 has been successfully used for fluorescence imaging in colon cancer SW480 cells.

Probing chromium(III) from chromium(VI) in cells by a fluorescent sensor

Cellular uptake of Cr(VI), followed by its reduction to Cr(III) with the formation of kinetically inert Cr(III) complexes, is a complex process. To better understand its physiological and pathological functions, efficient methods for the monitoring of Cr(VI) are desired. In this paper a selective fluorescent probe L, rhodamine hydrazide bearing a benzo[b]furan-2-carboxaldehyde group, was demonstrated as a red chemosensor for Cr(III) at about 586 nm. This probe has been used to probe Cr(III) which is reduced from Cr(VI) by reductants such as glutathione (GSH), vitamin C, cysteine (Cys), H2O2 and Dithiothreitol (DTT) by fluorescence spectra. Cr(VI) metabolism in vivo is primarily driven by Vc and GSH. Vc could reduce CrO4(2-) to Cr(III) in a faster rate than GSH. The indirectly detection limit for Cr(VI) by L+GSH system was determined to be 0.06 μM at pH=6.2. Moreover, the confocal microscopy image experiments indicated that Cr(VI) can be reduced to Cr(III) inside cells rapidly and the resulted Cr(III) can be captured and imaged timely by L.

Application of nanodiamonds in Cu(II)-based rhodamine B probes for NO detection and cell imaging

Nitric oxide functions as an important signalling molecule in many biological systems. Nanodiamonds (NDs) have attracted enormous attention in the field of biomaterial science for biosensing applications and drug/gene delivery. In this work, one novel Cu(ii)-based rhodamine B probe for NO detection was composed which was covalently bound to the nanodiamond (ND) surface. The results showed that the covalently conjugated ND probe could detect Cu2+ and NO sequentially with high sensitivity, high stability and good reusability compared to a free rhodamine B probe in CH3CN/HEPES (pH 6.8, v/v, 1 : 1). The detection limit for NO was estimated to be 10.5 nM. In addition, the response to NO was reversible in the presence of O3, which has attracted attention in the fields of biology and environmental science. Further analysis of confocal fluorescence microscopy images and the MTT assay indicated that the conjugated ND probe has favorable biocompatibility and low toxicity. This new Cu2+ and NO selective fluorescent ND probe may have potential applications in environmental science and biology.

Conformational stability of CopC and roles of residues Tyr79 and Trp83.

CopC is a periplasmic copper Chaperone protein that has a β‐barrel fold and two metal‐binding sites distinct for Cu(II) and Cu(I). In the article, four mutants (Y79F, Y79W, Y79WW83L, Y79WW83F) were obtained by site‐directed mutagenesis. The far‐UV CD spectra of the proteins were similar, suggesting that mutations did not bring any significant changes in secondary structures. Meanwhile the effects of mutations on the proteins function were manifested by Cu(II) binding. Fluorescence lifetime measurement and quenching of tryptophan fluorescence by acrylamide and KI showed that the microenvironment around Trp83 was more hydrophobic than that around Tyr79 in apoCopC. Unfolding experiments induced by guanidinium chloride (GdnHCl), urea provided the conformational stability of each protein. The Δ obtained using the model of structural elements was used to show the role of Tyr79 and Trp83. On the one hand, the induced by urea for Y79F, Y79W have a loss of 6.51, 2.03 kJ/mol, respectively, compared with apoCopC, proving that replacement of Tyr79 by Phe or Trp all decreased the protein stability, meaning that the hydrogen bonds interactions between Tyr79 and Thr75 played an important role in stabilizing apoCopC. On the other hand, the induced by urea for Y79WW83L have a loss of 11.44 kJ/mol, but for Y79WW83F did a raise of 1.82 kJ/mol compared with Y79W. The replacement of Trp83 by Phe and Leu yields opposite effects on protein stability, which suggested that the aromatic ring of Trp83 was important in maintaining the hydrophobic core of apoCopC.

Stability of proteins with multi-state unfolding behavior

A new model used to calculate the free energy change of protein unfolding is presented. In this model, proteins are considered to be composed of structural elements. The unfolding of a structural element obeys a two-state mechanism and the free energy change of the element can be obtained by a linear extrapolation method. If a protein consists of the same structural elements, its unfolding will displays a two-state process, and only the average structural element free energy change 〈ΔGelement0(H2O)〉 can be measured. If protein consists of completely different structural elements, its unfolding will show a multi-state behavior. When a protein consists of n structural elements its unfolding will shows a (n+1)-state behavior. A least-squares fitting can be used to analyze the contribution of each structural element to the protein and the free energy change of each structural element can be obtained by using linear extrapolation to zero denaturant concentration, not to the start of each transition. The measured ΔGprotein0(H2O) is the sum of the free energy change for each structural element. Using this new model, we can not only analyze the stability of various proteins with similar structure and similar molecular weight, which undergo multi-state unfolding processes, but also compare the stability of proteins with different structures and molecular weights using the average structural element free energy change 〈ΔGelement0(H2O)〉. Although this method cannot completely provide the exact free energy of proteins, it is better than current methods.

Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA

Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin-iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb(3+)) stoichiometry and an affinity of K=(2±-1)×10(11)M(-2) was determined. Pch-Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb(3+)) and Tb(3+) (acceptor). Pch-Tb binds to the Pch-Fe outer membrane transporter FptA with a dissociation constant (K(d)) of 4.6μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch.

Critical role of tyrosine 79 in the fluorescence resonance energy transfer and terbium(III)-dependent self-assembly of ciliate Euplotes octocarinatus centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins. It has been proven, using Tb3+ as a fluorescence probe, that EoCen has four calcium-binding sites. The sensitized emission arises from nonradiative energy transfer between the three tyrosine residues (Tyr46, Tyr72, and Tyr79) of the N-terminal half and the bound Tb3+ ions. To determine the most critical of the three tyrosine residues for the process of fluorescence resonance energy transfer, six mutants of the N-terminal domain of EoCen, which contain one (N-Tyr46/N-Tyr72/N-Tyr79) or two (N-Y46F/N-Y72F/N-Y79F) tyrosine residues, were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb3+ fluorescence of N-Y79F was most affected, displaying a 50% reduction compared with wild-type N-EoCen. Among the tyrosines, Tyr79 is the shortest mean distance from the protein-bound Tb3+ (at sites I/II), as calculated via the Förster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr79, which exists in a hydrophobic environment, has the highest quantum yield and a relatively long average lifetime. The decay of Tyr79 is the least heterogeneous among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr79 and the oxygen from the side chains of the residue Asn39. Kinetic experiments on tyrosine and Tb3+ fluorescence demonstrate that tyrosine fluorescence quenching is largely due to the self-assembly of EoCen, and that the quenching degrees of the mutants differ. Resonance light scattering and crosslinking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr79 also plays the most important role in the Tb3+-dependent self-assembly of EoCen among the three tyrosines.

Smart pH-responsive and high doxorubicin loading nanodiamond for in vivo selective targeting, imaging, and enhancement of anticancer therapy

Nanodiamond as a carrier for transporting chemotherapy drugs has emerged as a promising strategy for treating cancer. However, several factors have limited its extensive applications in biology, such as low drug loading, tending to aggregate and high drug loss under physiological conditions. In this work, to ensure a high drug capacity and low drug leakage in blood circulation, and especially to ensure that it is delivered to the tumor region, a smart pH-responsive drug delivery system is designed and prepared using doxorubicin (DOX) adsorbed onto PEGylated nanodiamond in sodium citrate medium (ND-PEG-DOX/Na3Cit, NPDC). The system can significantly enhance cellular uptake to exert a therapeutic effect in comparison to the free drug. And more importantly, DOX was released in a sustained and pH-dependent manner, exhibiting excellent stability under physiological conditions. In addition, NPDC could enter into cells via both the clathrin- and caveolae-mediated endocytosis pathways, and then the dissociated DOX migrated into the nucleus to block the growth of cancer cells. Furthermore, NPDC can significantly inhibit cell migration and change the cell cycle. Excitingly, the NPDC system was very smart for the effective enrichment at the tumor site in vivo and enhancing antitumor efficiency with low toxicity beyond conventional DOX treatment in cancer cells and a nude mouse model. So this study introduces a simple and effective strategy to design a promising drug delivery platform for improving the biomedical applications of the smart nanodiamond carriers.