Peptidyl-Resin Substrates as a Tool in the Analysis of Caspase Activity

Caspases, proteolytic enzymes belonging to the group of cysteine proteases, play a crucial role in apoptosis. Understanding their activity and substrate specificity is extremely important. Fluorescence-based approaches, including fluorogenic substrates, are generally used to confirm cleavage preferences. Here we present a new method of substrate specificity and activity analysis based on the application of fix-charge tagged peptides located on the resin. The proteolysis of peptide bond on the resin, occurring even with low efficiency, results in the formation of N-terminal fragments of model peptide containing ionization enhancers in the form of quaternary ammonium groups, allowing for ultrasensitive and reliable analysis by LC-MS/MS. The possibility of application of the proposed solution was tested through the analysis of substrate specificity and activity of caspase 3 or 7. The obtained results confirm the known substrate specificity of executioner caspases. Our solution also allowed us to observe that caspases can hydrolyze peptides shorter than those presented to date in the scientific literature.     

基于氨基酸位置特异性的蛋白质Loop区结构预测改进方法

蛋白质loop区的结构预测是理解蛋白质功能的重要一环,而长loop区的结构预测至今还是生物信息学中的难题.目前己经出现了多种loop结构的算法,其中LEAP是预测精度最高的算法之一,但它在长loop区初始主链构象采样上仍有较大的改进余地.本文中我们将蛋白质二级结构预测算法SPINE X与LEAP算法结合起来,构建了新的主链扭转角分布图(拉氏图),在主链初始构象采样中引入氨基酸在蛋白序列中的位置特异性信息,使得初始构象的采样更具针对性,对取自CASP10单链蛋白的loop测试集的分析表明,对长度为10,11,12个氨基酸的长loop区,改进后算法都比原始LEAP算法的预测精度有显著提升.这种引入氨基酸位置特异性从而提高预测精度的思路有望进一步推广至loop结构预测的其他算法.     

Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.     

头发铅含量测定的应用价值评价

调查了125例2－5岁儿童铅中毒状况，测定其全血铅含量和头发铅含量，以全血铅含量为标准，应用诊断试验评价方法评价头发铅测定的应用价值。结果表明，该试验的灵敏度可达88．3％，应用串联试验，即同时测定头发中锌、铁、钙含量，可将该试验的特异度提高至81．8％，阳性预测值达69．6％，阴性预测值达78．8％。     

Dendrimers and combinatorial chemistry—tools for fluorescent enhancement in protease assays

FRET based systems are some of the best methods available to detect and monitor proteolytic activity. To enhance fluorescent signals and hence assay sensitivity, two different systems were developed using two different dendrimeric constructs. In the first case, a triple branched dendrimer bearing three dansyl groups was used to enhance assay sensitivity and showed a significant enhancement of fluorescence following enzymatic cleavage. In another example, a tris-fluorescein probe, that undergoes self-quenching, was utilized in a combinatorial library synthesis to map the substrate specificity of proteases.     

A pseudoreceptor modelling study of the varicella-zoster virus and human thymidine kinase binding sites

Summary A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV), thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.     

Laccase Encapsulation in ZIF-8 Metal-Organic Framework Shows Stability Enhancement and Substrate Selectivity

CgL1 laccase from Corynebacterium glutamicum was encapsulated into the metal-organic framework (MOF) ZIF-8 which was synthesized in a rapid enzyme friendly aqueous synthesis, the fastest in situ encapsulation of laccases reported to date. The obtained enzyme/MOF, i. e. laccase@ZIF-8 composite showed enhanced thermal (up to 70 °C) and chemical (N,N-dimethylformamide) stability, resulting in a stable heterogenous catalyst, suitable for high temperature reactions in organic solvents. Furthermore, the defined structure of ZIF-8 produced a size selective substrate specificity, so that substrates larger than the pore size were not accepted. Thereby, 2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was used to verify that the enzyme is immobilized inside the MOF versus the outside surface. The enzyme@MOF composite was analyzed by atomic absorption spectroscopy (ASS) to precisely determine the enzyme loading to 2.1 wt%.     

Aminolysis ofO-alkylO-4-nitropyenyl chloromethylphosphonates in reverse micellar solutions of the nonionic surfactant

The reaction ofn-hexylamine withO-alkylO-4-nitrophenyl chloromethylphosphonates in toluene solutions of poly(ethylene glycol)-600 monolaurate (PM) has been studied by spectrophotometry. The reverse micelles of the nonionic surfactant increase more than tenfold the observed rate constant of aminolysis. The catalytic activity of the surfactant is practically independent of the alkyl radical length of phosphonate. An increase in the concentration of amine results in a decrease in the catalytic efficiency. The character of the dependence of the rate constant on the concentration of PM is affected by the alkyl chain length of the substrate. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 9, pp. 1735–1738, September, 1998.     

The use of a radiation inactivation method for characterization of the structure and catalytic properties of enzymes

The use of a radiation inactivation method for studies of the properties of enzymes of different classes is discussed. The possibilities of characterization of enzymes on the basis of the radiation-chemical parameters concerned are described.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No, 12, pp. 2828–2836, December, 1996.     

A proposed complementary pairing mode between single-stranded nucleic acids and β-stranded peptides: A possible pathway for generating complex biological molecules

A structure for a heterologous double-stranded molecule consisting of an oligoribonucleotide and a β-stranded oligopeptide, paired through complementary Watson-Crick-type hydrogen bonding, is proposed. The basis for such complementary pairings between oligoribonucleotides and oligopeptides is the close correspondence of the distances between the side-chains attached to the backbones of the two molecules. Both inter-nucleotide spacing and inter-amino acid side-chain spacing are approximately 3.4 Å. These kinds of interactions may have implications in protein-mediated-RNA splicing. Formation of a heterologous duplex through such a pairing mode could provide a simple coding mechanism for a reciprocal information transfer between oligonucleotides and oligopeptides. Because of its simplicity and versatility, allowing both specific and nonspecific coding, interactions via these kinds of pairing may have been relevant for prebiotic molecular evolution and for generating complex biological molecules.