Single-Molecule FRET of Membrane Transport Proteins

Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane-embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high-resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state-of-the-art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design.     

Mechanism of Reaction of Proteins with Reactive Dyes:III—Reactivity of Soluble Proteins with Chlorotriazine Dyes

The reactivities of ten soluble proteins with a monochlorotriazine reactive dye were measured under various conditions and compared with those of mixtures of simple model compounds representing the reactive groups of the proteins. Both sets of results agreed wellwith reactivity curves calculated from measurements of the individual model compounds. The mechanisms of the reactions between the dye and proteins are discussed with reference to the dyeing of wool and other proteinaceous fibres.     

Mechanism of Reaction of Proteins with Reactive Dyes IV—Reactivity of Wool with Chlorotriazine Dyes*

Chlorinated wool was dyed with a monochlorotriazine reactive dye under infinite dyebath conditions. The kinetics of dyeing conformed to a composite equation derived from the Danckwerts and Hill equations for diffusion into an infinite cylinder with surface saturation. The rate constants for the reaction between the dye and the fibre obtained from this equation agreed well with theoretical constants calculated from the reactivities of model compounds and data obtained from amino-acid analysis. The relative contributions of the individual types of reactive site to the total reactivity of wool are discussed.     

Mechanism of Reaction of Proteins with Reactive Dyes:I-Literature Survey

The chemistry of reactive dyes and wool keratin is described briefly. Published investigations of the mechanisms of dyeing of wool and other protein fibres with reactive dyes are discussed and compared.     

Mechanism of Reaction of Proteins with Reactive Dyes:II—Reactivity of Simple Model Compounds with Chlorotriazine Dyes

The chemistry of the functional groups in wool is described and the selection of appropriate model compounds for them is discussed. The measurement of the reactivities of these model compounds and possible mechanisms of their reactions are discussed. The nucleophilic anion is the important species in the reaction of a chlorotriazine dye with the thiol group in cysteine or the hydroxyl and phenolic groups in serine, threonine and tyrosineresidues. The reaction of a chlorotriazine dye with the amino groups of lysine and the N-terminal amino acids, with the imidazole group of histidine and with the guanidinogroup of arginine takes place mainly with the unprotonated base. Evidence in support of these mechanisms is provided by an investigation of the reactions of the dye with aqueous ammonia and anhydrous liquid ammonia.     

Peanut FAD2 Genotype and Growing Location Interactions Significantly Affect the Level of Oleic Acid in Seeds

The level of oleic acid is an important parameter in determining seed nutritional quality and oil stability. The level of oleic acid in peanut is genetically controlled by a pair of fatty acid desaturase genes (FAD2A and FAD2B), but the environmental conditions of the production sites can also have a significant effect. To investigate the effect of gene and environment interaction, 45 accessions were grown at three locations for 2 years. Environmental data were collected; individual plants were genotyped with functional SNP markers from FAD2A and FAD2B; and seed level of oleic acid was determined by gas chromatography. Three FAD2A/FAD2B genotypes (448G/no insertion 442A, 448A/no insertion 442A, and 448A/insertion 442A) were identified and designated as G/W, A/W, and A/A, respectively. A/A genotype averaged the highest level of oleic acid (80.0%), followed by A/W (56.0%), and then G/W (40.7%). Analysis of gene and environment interaction revealed that oleic acid phenotype plasticity could be explained by the interaction of FAD2 genotype and photothermal time, which quantified environmental conditions. The A/W genotype was the most sensitive to photothermal time changes. The oleic acid plasticity revealed in this study would be useful for breeders, farmers, and product processors.     

Site‐Specific Labelling of Native Mammalian Proteins for Single‐Molecule FRET Measurements

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single‐molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site‐specific coupling of organic dyes into native, non‐recombinant mammalian proteins is lacking. We address this issue showing the site‐specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post‐translationally modified proteins on the single‐molecule level, among them the hitherto inaccessible human Argonaute 2.     

Photoreactive Dyes: The Potential of Azido-substituted Dyes as Latent Reactive Dyes for Wool

Dyes containing azido substituents have been examined as latent reactive dyes for wool and nylon. Three different substituents, viz. azidophenyl, azidosulphonyl and diazidotriazinyl, were used. It was found that dyes containing azidophenyl groups could be applied under conditions where little reaction with the fibre occurred. These dyes could then be induced to react covalently with the fibre either by irradiation with light or by ironing at 200d?C. However, the binding of the dye to the fibre occurs in rather low yield (30–40%) and, in some cases, is accompanied by a marked colour change. Dyes containing azidosulphonyl or diazidotriazinyl substituents react with wool and nylon during application, and they are therefore unsuitable as latent reactive dyes.     

Analysis of SAXS data from ionomer systems

A model was proposed earlier for the microstructure of ionomers which attributes the small-angle X-ray scattering (SAXS) peak observed in these materials to interparticle interference between small ionic domains arranged in the hydrocarbon matrix with a liquid-like degree of order. In this work, that model is used to interpret SAXS results obtained for a number of different ionomer systems. In addition, the effect of swelling of sulphonated polystyrene ionomers with water is investigated using this approach. Finally, the effect of temperature on the scattering was studied. The results reveal some interesting differences in domain size between different ionomer systems. The ethylene/methacrylic acid ionomers contain very small domains consistent with the concept of a multiplet, while the sulphonated EPDM rubbers have rather large domains. The results of the water absorption studies were inconsistent with the assumption that there is no change in the number of ionic domains upon swelling but if that assumption is not made, the data can be rationalized with the model. The elevated temperature measurements demonstrated the stability of the ionic domains even at temperatures well above those where the materials can be processed, leading to speculation regarding the mechanism of melt flow in ionomers.     

现代活性染料与分散染料的发展

近年分散染料与活性染料的发展因受相应纤维大幅增长的关联,产量和品种开发均列第一、二位.本文就这二类染料进行详细评述.近十年活性染料的新发色母体和活性基没有新的突破性进展,引入取代基改善染色性能的结构修饰和商品化技术成为活性染料发展重点.分散染料则以开发杂环型分散染料为重点.本文所列染料分子结构均为近年开发的品种,许多是鲜为人知的.     

酸性染料和中性染料的重金属问题

酸性染料是一类重要的染料，广泛用于羊毛、丝绸、锦纶和皮革的染色，其中一些深色酸性金属络舍染料包括中性染料，具有深浓色调、着色强度高、耐光、耐气候和湿牢度好的优点，但因含有有害重金属，对人体健康和环境造成严重危害，受到环保法规的限用或禁用。本文对这类染料的大部分品种进行曝光，以使其避免被选用，并就酸性金属络合染料代用品的重要品种加以评述。     

Assessment of branching in polymers from small angle X-ray scattering (SAXS): SAXS from model comb-branched polystyrenes

Comb-branched polystyrenes having both fixed backbone length and number of branches but variable branch lengths were prepared anionically. Small angle X-ray scattering of the model polystyrenes from methyl ethyl ketone (MEK) solutions at 25°C was examined and it is shown that the mass per unit length and cross-sectional radius of gyration increase with increasing branch length. A maximum appears in the Kratky plot for the branched polymers. While the linear polymer displays an essentially Gaussian segment distribution in solution, the branched polymers deviate considerably from the behaviour of the linear polymer under the same conditions.     

酸性染料和中性染料的重金属问题

酸性染料是一类重要的染料,广泛用于羊毛、丝绸、锦纶和皮革的染色,其中一些深色酸性金属络合染料包括中性染料,具有深浓色调、着色强度高、耐光、耐气候和湿牢度好的优点,但因含有有害重金属,对人体健康和环境造成严重危害,受到环保法规的限用或禁用.本文对这类染料的大部分品种进行曝光,以使其避免被选用,并就酸性金属络合染料代用品的重要品种加以评述.     

SAXS方法及其在多孔材料研究中的应用

小角X射线散射（SAXS）是一种有效，重要的亚微结构分析手段。本文主要介绍了SAXS对于非理想两相体系的解析方法及其在多孔材料中的应用。     

Quantitative Analysis of Tau-Microtubule Interaction Using FRET

The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.     

荧光染料概述

荧光染料是近年发展起来的一类很有前景的功能性染料。本文简要概述了荧光染料的发展。以香豆素及其衍生物为主,介绍了几类不同荧光染料的合成方法。     

菁染料的J-聚集体

综述了近年来有关菁染料J-聚集体的研究。     

活性染料和直接染料的生态毒理特性分析

本文从化学结构和应用两个方面详细地分析了纤维素纤维用活性染料和直接染料的生态毒理特性,指出活性染料是一类很有发展前途的染料,它对环保型染料和绿色纺织品的发展具有重要的参考价值.