Interaction of zinc finger ZNF191(243—368) and its I323 W and R327W mutants with oligonucleotides by florescence spectrum

Two mutants of the zinc finger protein, ZNF191 (243–368) I323W and R327W, are successfully obtained by site-directed mutagenesis. The fluorescence spectrum is used to study the interaction between these two mutants and the oligonucleotides. The influence of the mutation on the interaction has been studied using ethidium bromide (EB) as the fluorescence probe. The binding constants of the I323W-DNA and R327W-DNA have been calculated and the possible binding models have been discussed.     

Expression and Purification of ZNF191（243-368） in Three Expression Systems

ZNF191 （243-368）, a new human zinc finger protein, probably relates to some hereditary diseases and cancers, To obtain adequate amount of ZNF191（243-368） for the study of its property, structure and function, three different expression systems of inclusion-body, glutathione S-transferase （GST）, and hexahistidine （6 × His） were used and compared. Among these systems, the expression level of ZNFI91（243-368） was increased in inclusion body system under a higher isopropylthio-β-D-galactoside （IPTG） concentration, but the non-target proteins were also increased more, which made its purification more difficult and the yield lower. The expression of His-tag fusion protein was almost not affected by IPTG concentration, temperature and inducing time. At a high IPTG concentration the highest expression yield for GST fusion protein was obtained. And the fusion proteins can be partially purified by a single affinity chromatography step. The fusion protein systems show advantages for expression of these proteins.     

一个新锌指蛋白的表达和纯化--ZNF191蛋白及其锌指区ZNF191(243-368)

通过PCR方法 ,将ZNF1 91基因的cDNA的第一开链阅读框 (ORF)及其锌指区ZNF1 91 ( 2 43 36 8)装入PTSA 1 8表达载体 ,以E .coliBL2 1 (DE3)为宿主菌 ,成功地表达了目标蛋白 ,并通过DEAE5 2 ,CM2 3,Heparin Sephrose 4B等层析柱对目标蛋白进行了纯化 .ZNF1 91锌指蛋白及其锌指区ZNF1 91 ( 2 43 36 8)蛋白经考马斯亮蓝染色的聚丙烯酰胺凝胶电泳检查 ,纯度达单一带水平 .其中对ZNF1 91蛋白经N端氨基酸微序列分析证明为目的蛋白 .     

锌指蛋白ZNF191(243~368)突变体I323W和R327W与寡聚核苷酸作用的荧光光谱

利用PCR方法对锌指蛋白ZNF191(243~368)的Ile323和Arg327进行定点突变, 成功地获得了突变体蛋白ZNF191(243~368) I323W和R327W; 并利用荧光光谱研究了锌指蛋白和它的突变体蛋白ZNF191(243~368)I323W和ZNF191(243~368) R327W与寡聚核苷酸的相互作用. 以溴化乙锭(EB)为荧光探针, 考察了I323W和R327W的突变对锌指蛋白ZNF191(243~368)结合寡聚核苷酸的影响.并计算了锌指蛋白突变体I323W和R327W与DNA的结合常数K_Z. 讨论了可能的结合模式.     

Role of protein structure and the role of individual fingers in zinc finger protein–DNA recognition: a molecular dynamics simulation study and free energy calculations

Molecular dynamics and MM_GBSA energy calculations on various zinc finger proteins containing three and four fingers bound to their target DNA gave insights into the role of each finger in the DNA binding process as part of the protein structure. The wild type Zif 268 (PDB code: 1AAY) gave a ΔG value of ??76.1 (14) kcal/mol. Zinc fingers ZF1, ZF2 and ZF3 were mutated in one experiment and in another experiment one finger was cut and the rest of the protein was studied for binding. The ΔΔG values for the Zinc Finger protein with both ZF1 and ZF2 mutated was +?80 kcal/mol, while mutating only ZF1 the ΔΔG value was +?52 kcal/mol (relative to the wild type). Cutting ZF3 and studying the protein consisting only of ZF1 linked to ZF2 gave a ΔΔG value of +?68 kcal/mol. Upon cutting ZF1, the resulting ZF2 linked to ZF3 protein gave a ΔΔG value of +?41 kcal/mol. The above results shed light on the importance of each finger in the binding process, especially the role of ZF1 as the anchoring finger followed in importance by ZF2 and ZF3. The energy difference between the binding of the wild type protein Zif268 (1AAY) and that for individual finger binding to DNA according to the formula: ΔΔG_{linkers, otherstructuralfactors}?=?ΔG_zif268???(ΔG_F1+F2+F3) gave a value?=???44.5 kcal/mol. This stabilization can be attributed to the contribution of linkers and other structural factors in the intact protein in the DNA binding process. DNA binding energies of variant proteins of the wild type Zif268 which differ in their ZF1 amino acid sequence gave evidence of a good relationship between binding energy and recognition and specificity, this finding confirms the reported vital role of ZF1 in the ZF protein scanning and anchoring to the target DNA sequence. The role of hydrogen bonds in both specific and nonspecific amino acid-DNA contacts is discussed in relation to mutations. The binding energies of variant Zinc Finger proteins confirmed the role of ZF1 in the recognition, specificity and anchoring of the zinc finger protein to DNA.     

A novel and stereoselective synthesis of (±)-cephalotaxine and its analogue

Cephalotaxine (1) and its analogue (3) were stereoselectively synthesized from proline via the pyrrolobenzazepine (4) through Claisen rearrangement, cationic cyclization, and regio- and stereo-selective hydroxylation.     

Synthesis of a novel perylene diimide derivative and its charge transfer interaction with C_(60)

A novel organic electron acceptor, N,N′-dipyrimidinyl-3,4,9,10-perylene-tetracarboxylic diimide (DMP), was designed and synthesized. The molecular structure was characterized by FTIR spectrum and elemental analysis. By cyclic voltammetry measurements, DMP was found to possess a lower LUMO energy level than N,N′-diphenyl-3,4,9,10-perylene-tetracarboxylic diimide due to the stronger electron-withdrawing pyrimidinyl group than the phenyl group. Fluorescence quenching is observed in a dual-layer film consisting of a DMP layer and a C60 layer and was attributed to the charge transfer at the interface due to the energy level offset between DMP and C60.     

Evaluation of pH-sensitive poly(β-amino ester)-graft-poly(ethylene glycol) and its usefulness as a pH-sensor and protein carrier

In this study, some possible biomedical applications of a pH-sensitive and amphiphilic copolymer as a pH sensor and protein delivery system are reported. PAE-g-PEG was used as a pH-sensitive polymer that can exhibit a sharp pH-dependent transition. Various fluorescent dyes including pyrene and RITC can be used to label the pH-sensitive polymer PAE-g-PEG, which was evaluated for protein encapsulation. pH-sensing was possible by observing excimer formation of the labeled pyrene via pH-dependent expansion of the polymeric chain. Also, it was confirmed that FITC-BSA could be entrapped in RITC-labeled pH-sensitive micelles of PAE-g-PEG by FRET. As a result, PAE-g-PEG can be a pH sensor and carrier for protein delivery.     

Synthesis and pharmacological activity of a silicon—zinc—boron-containing glycerohydrogel

Russian Chemical Bulletin - A new pharmacologically active nanostructured silicon—zinc—boron-containing glycerohydrogel was synthesized by the sol—gel method using silicon, zinc,...     

Metal complexes of a novel bis-β-diketone-type ligand and its copper(II) complexes of two-photon biological imaging

A curcumin derivative ligand,1,7-bis(3-methoxyl-4-acetoxyl)phenyl-1,6-heptadiene-3,5-diketone (diacetylcurcumin,abbreviated as HL),and its Cu and Ni complexes have been synthesized and fully characterized by elemental analyses,IR,1 HNMR and molar conductivity.The resulting complexes exhibit two-photon excited fluorescence (TPEF) in DMF,and have been proven to be potentially useful for two-photon microscopy imaging in living cells.In addition,cytotoxicity tests showed that the low-micromolar concentrations of ML 2 did not cause significant reduction in cell viability over a period of at least 24 h and should be safe for further biological studies.     

Development of flexible zinc–air battery with nanocomposite electrodes and a novel separator

In this paper, we present the development of flexible zinc–air battery. Multiwalled carbon nanotubes(MWCNTs) were added into electrodes to improve their performance. It was found that MWCNTs were effective conductive additive in anode as they bridged the zinc particles. Poly(3,4-ethylenedioxythiophene)polystyrene sulfonate(PEDOT:PSS) was applied as a co-binder to enhance both the conductivity and flexibility. A poly(acrylic acid)(PAA) and polyvinyl alcohol(PVA) coated paper separator was used to enhance the battery performance where the PVP–PAA layer facilitated electrolyte storage. The batteries remained functional under bending conditions and after bending. Multiple design optimizations were also carried out for storage and performance purposes.     

Vibrational spectra of carboxylato complexes—I. Infrared and Raman spectra of beryllium(II) acetate and formate and of zinc(II) acetate and zinc(II) acetate dihydrate

The spectra of the title compounds have been recorded and interpreted. On this basis, the structures of the anhydrous compounds Be(OOCH)₂, Be(OOCMe)₂, Zn(OOCMe)₂ are predicted to have tetrahedral coordination with the carboxylate ions acting as bidentate ligands.     

Synthesis of a novel zwitterionic biodegradable poly (α,β-L-aspartic acid) derivative with some L-histidine side-residues and its resistance to non-specific protein adsorption

A novel zwitterionic polypeptide derivative, denoted as His-PAsp/PAsp, was successfully synthesized by amidation of Poly (α,β-L-aspartic acid) with L-histidine methyl ester. Turbidity, zeta potential and 1H NMR measurements were used to study the aggregation behaviors of His-PAsp/PAsp under different pH values. The modified polypeptide derivative composed of electro-negatively carboxylic and electro-positively imidazole residues randomly so as to bear opposite charges at pH values above or below the isoelectric point. When the zwitterionic polypeptide was coated on silicon wafer as a model substrate material, the absorption resistance of fibrinogen, a blood protein resulting in the blood coagulation cascade, on the coated surface was measured. It was found that the adsorption amount of fibrinogen on the polypeptide-coated surface depended on the dose of the polypeptide on silicon wafer. Obvious resistance of the fibrinogen adsorption on the polypeptide-coated surface was observed. Since its good biodegradability and superior anti-protein-fouling property, this pH-responsive zwitterionic polypeptide is a promising candidate for surface modification in many biomedical applications, including medical implants, drug delivery carriers, and biosensors.     

Proton-Template Synthesis of a П─Conjugated Macrocycle and Structure of its Ni_2(Ⅱ)—Complex

Robson-typemacrocycliccomplexesareplanarbinuclearcomplexesderivedfromSchiff'sbasecondensation0f2,6-diformyl-4-alkylphenolwithalkylenediamineinthepresenceofmetal;".Inthepastmorethan2Oyearsal0tofsuchcomplexesweresgnthesisedinthiswayandinvestigated':'>.Butthecomplexesofsuchamacrocyc1ewith1,2-phenylenediaminebridgewereneverrePOrteduntilveryrecentlyBrychcyetal.havegotsomeofthemalsobymetal-templatemethed`&,J.Sinceall-ring-systemoftheligandisconugated,itScomplexeshavemoreinterestfortheirexpectedun…     

Photoinduced reactivity and cytotoxicity of a series of zinc(II)–flavonolate derivative complexes

Transition Metal Chemistry - A series of Zn(II)-based complexes as photoinduced carbon-monoxide-releasing molecules (photoCORMs), viz. (L)Zn(R)]ClO4 with...     

Synthesis and spectral characterization of zinc(II) complexes of N(4)-substituted thiosemicarbazone derived from salicylaldehyde: Structural study of a novel –OH free Zn(II) complex

Five novel zinc(II) complexes of salicylaldehyde 3-hexamethyleneiminyl thiosemicarbazone were synthesized and physico-chemically characterized. The complexes were given the formulae [Zn(HL)₂] (1), [Zn(L)py] (2), [Zn(L)bipy] · 3.5H₂O (3), [Zn(L)phen] · H₂O (4) and [Zn(L)γ-pic] (5). The thiosemicarbazone binds the metal as a dianionic ONS donor ligand in all the complexes, except in 1. Compound 1 is a homoleptic complex with zinc occupying the center of a distorted tetrahedral environment and being coordinated by two azomethine nitrogen and two thiolate sulfur atoms.     

Synthesis and spectroscopic properties of a novel zinc phthalocyanine with four 17-membered crown ether voids

A novel zinc phthalocyanine containing four 17-crown-5 ether voids(17C5ZnPc) has been synthesized and characterized. UV-visible absorption and fluorescence emission spectra and as-sociated photophysical parameters have been determined. In contrast to most of the crown ether substituted phthalocyanines, no cofacial dimer formation is observed in the presence of alkali metal salts. In addition to the fluorescence at 710 nm from S1, a strong upper excited state (Soret 52) emission around 424 nm has been detected for the first time in the phthalocyanine series. Fluorescence decay of S1 and S2 emission can be analyzed by mono- and biexponential fits respectively. X-ray structure analysis showed that the crown ether unit is conformationally deformed and oblate that may account for the unusual spectroscopic properties.     

Preparation of a novel cyclodextrin derivative of benzimido-β-cyclodextrin and its enantioseparation performance in HPLC

A novel cyclodextrin (CD) derivative, mono-6-deoxy-benzimide-β-CD (MB-β-CD), in which a rigid substituent was linked to the narrow edge of the CD with a flexible H(2)C-N group, was successfully synthesized through the condensation of mono-6-deoxy-6-amino-β-cyclodextrin and benzaldehyde. To evaluate its enantioseparation abilities and investigate the role of the CD substituents and linkage in chiral recognition, MB-β-CD and mono-6-deoxyphenylimine-β-CD (MP-β-CD) with a rigid linkage were compared in the separation of 36 chiral compounds in a methanol/water mobile phase. The separation results showed that most of the analytes with rigid structures afforded better enantioresolutions on the MP-β-CD (with a rigid linkage) chiral stationary phase (CSP), while better enantioseparations for analytes with flexible structures and big steric hindrance were obtained on the MB-β-CD (with a flexible linkage) CSP. The former exhibited a specificity for the analyte structures, while the latter was more adaptable. Molecular dynamics simulations were performed to further understand the discrimination process and the function of the CD side arm.