Monomer formation and function of p-hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide/water mixtures

It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric forms of PHBH could be observed separately by varying the size of the micelles. A considerable decrease in the K(M) value for p-hydroxybenzoate (POHB) was found for monomeric PHBH, accompanied by a 1.5-fold decrease in enzymatic activity. The same tendency was observed when monomers of PHBH were formed by adding DMSO to the buffer. The FAD in PHBH and PHBH labeled with the fluorescence dye Alexa488 was investigated by time-resolved fluorescence anisotropy to observe monomer formation in water/DMSO mixtures. Monomer formation of PHBH occurred gradually with increasing DMSO content in the mixture. Pure PHBH monomers were detected at DMSO concentrations of 30 % (v/v) and higher.     

Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers

For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild‐type monomer and an inactive monomer in which an active‐site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.     

Site-specific labeling of proteins by using biotin protein ligase conjugated with fluorophores

Biotin protein ligase (BPL) mediates the covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP). This biotinylation in Sulfolobus tokodaii is unique in that BPL forms a tight complex with the product, biotinylated BCCP, and this property was exploited for fluorescent labeling of a membrane protein. Thus, the truncated form of BCCP (BCCPΔ100, 69 residues) was fused to either the N or C terminus of the bradykinin B2 receptor (B2R). The resulting fusion proteins, BCCPΔ100-B2R and B2R-BCCPΔ100, respectively, were separately expressed in mammalian HEK293 cells, and labeled with BPL conjugated with a fluorophore: either fluorescein, DyLight549 or green fluorescent protein. The fusion proteins were biotinylated and bound to BPL, thereby giving rise to strong fluorescence along the periphery of the cell. Some were capable of binding bradykinin and an antagonist. When stimulated with the former, the receptor translocated to the cytosol; this suggests that the labeled receptor retains its integrity in terms of ligand-binding and translocation.     

Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.     

Fluorescence Properties of Flavin Semiquinone Radicals in Nitronate Monooxygenase

Fluorescent cofactors like flavins can be exploited to probe their local environment with spatial and temporal resolution. Although the fluorescence properties of the oxidized and two-electron-reduced states of flavins have been studied extensively, this is not the case for the one-electron-reduced state. Both the neutral and anionic semiquinones have proven particularly challenging to examine, as they are unstable in solution and are transient, short-lived species in many catalytic cycles. Here, we report that the nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 is capable of stabilizing both semiquinone forms anaerobically for hours, thus enabling us to study their spectroscopy in a constant protein environment. We found that in the active site of NMO, the anionic semiquinone exhibits no fluorescence, whereas the neutral semiquinone radical shows a relatively strong fluorescence, with a behavior that violates the Kasha–Vavilov rule. These fluorescence properties are discussed in the context of time-dependent density functional theory calculations, which reveal low-lying dark states in both systems.     

Tunable photoactivation of a post-translationally modified signaling protein and its unmodified counterpart in live cells

An ideal technology for direct imaging of post-translationally modified proteins would be one in which the appearance of a fluorescent signal is linked to a modification dependent protein-activation event. Herein, we utilize the protein semisynthesis technique, expressed protein ligation (EPL), to prepare caged analogues of the signaling protein Smad2; the function and fluorescence of the analogues were then photocontrolled in a correlated fashion. We show that this strategy permits titration of the cellular levels of active phosphorylated Smad2 in its biologically relevant, full-length form. We also prepared a nonphosphorylated, caged full-length Smad2 analogue labeled with an orthogonal fluorophore, and simultaneously imaged the phosphorylated and nonphosphorylated forms of the protein in the same cell. This strategy should enable the dissection of the cellular consequences of post-translational modifications (PTMs) by direct comparison of the behavior of the modified and unmodified forms of the protein following uncaging.     

Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.     

Interaction of human serum album and c(60) aggregates in solution

An important property of C(60) in aquatic ecotoxicology is that it can form stable aggregates with nanoscale dimensions, namely nC(60). Aggregation allows fullerenes to remain suspended for a long time, and the reactivity of individual C(60) is substantially altered in this aggregate form. Herein, we investigated the interaction of nC(60) and human serum album (HSA) using the methods of fluorescence, fluorescence dynamics, circular dichroism (CD), and site marker competitive experiments. We proposed a binding model consistent with the available experimental results for the interactions of nC(60) with HSA. During the interaction process, the structure and conformation of HSA were affected, leading to functional changes of drug binding sites of HSA.     

2－芳基苯并噁唑的荧光猝灭和光诱导电子转移研究

本工作对两种不同的２－芳基苯并　唑化合物溶液荧光被四氯化碳所猝灭的机理进行了详细研究，通过多种途径研究表明该猝灭过程具有光诱导电子转移性质，工作还利用此电子转移所形成的活泼自由基来引发烯类单体的聚合，得到了有一定聚合度的聚甲基丙烯酸甲酯。     

Lead–yeast hexokinase interaction: modifications to protein structure caused by the metal

The interaction between lead and yeast hexokinase has been studied. Lead provokes a large variation in the aggregation state of the protein, forming bigger structures of high molecular mass. This phenomenon is characterized by a small modification in the tridimensional structure and a great variation in the secondary structure. There is a loss in α‐helix which is compensated by an enhancement in β‐sheet. The polypeptide chain is more stable in the β‐sheet structure corresponding to the aggregate forms. During this change the enzyme maintains a high level of activity in the monomer and also in the aggregate form. This implies that the enzyme function is not greatly affected by the change, and active sites are retained without important modifications. According to kinetic measurements the ATP site is more affected than the glucose site. There is a mixed type inhibition with a main competitive component when glucose acts as a variable substrate. © 2001 Society of Chemical Industry     

Decay Lifetimes of Para-Substituted Polystyrene in Solid Films and in Dichloromethane

The fluorescence decay times of polystyrene, poly(4-bromostyrene), poly(4-chlorostyrene), poly(4-methylstyrene), poly(α-methylstyrene), poly(4-methoxystyrene), and poly(4-tert butylstyrene) were measured in solid films and in dichloromethane solution. A detailed analysis of the emission profile performed by nanosecond time resolved fluorescence spectroscopy confirmed the presence of monomer fluorescence as well as excimer fluorescence in both media. Monomer fluorescence decay times gave shorter times compared with that of excimer decay times in dichloromethane solution. Fluorescence from substituted polystyrene was mainly excimer fluorescence with shift of maximum emission to longer wavelength. The ratio of monomer to excimer contributions was found to be dependent on the emission wavelength, but was not affected by polymer concentration. Both monomer and excimer fluorescence lifetimes as well as excimer intensity increase with increasing emission wavelength. An accompanying decrease in monomer contribution is also observed is solution in comparison with that in solid films.     

A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein that forms an α-helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding-induced structural changes are low-throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.     

吖啶类荧光探针与蛋白质相互作用的研究

文章研究3种新型吖啶类荧光探针(N-(2-二甲氨基)乙基-9-氯吖啶-4-甲酰胺(NCAF)、9-[(N-2-二甲氨乙基)吖啶-4-甲酰胺]-α-丙氨酸(NAFA)和4,9-二[N-(2-二甲氨基)乙基]-9-吖啶胺-4-甲酰胺(DNAF))与牛血清白蛋白(BSA)的相互结合作用机理。分别对3种吖啶类探针自聚集情况、与牛血清白蛋白结合常数KA和结合位点数n、热力学参数H、G以及S、能量转移效率E和结合距离R0进行比较,并对实验数据进行分析。研究了3种吖啶类探针对蛋白质内源荧光猝灭的猝灭机制和主要作用力类型,为进一步研究新的生物探针及其在生物大分子识别分析应用提供了一定的实验和理论支持。     

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on F?rster Resonance Energy Transfer Techniques

Förster resonance energy transfer (FRET) occurs when the distance between a donor fluorophore and an acceptor is within 10 nm, and its application often necessitates fluorescent labeling of biological targets. However, covalent modification of biomolecules can inadvertently give rise to conformational and/or functional changes. This review describes the application of intrinsic protein fluorescence, predominantly derived from tryptophan (λ_EX ∼ 280 nm, λ_EM ∼ 350 nm), in protein-related research and mainly focuses on label-free FRET techniques. In terms of wavelength and intensity, tryptophan fluorescence is strongly influenced by its (or the protein’s) local environment, which, in addition to fluorescence quenching, has been applied to study protein conformational changes. Intrinsic Förster resonance energy transfer (iFRET), a recently developed technique, utilizes the intrinsic fluorescence of tryptophan in conjunction with target-specific fluorescent probes as FRET donors and acceptors, respectively, for real time detection of native proteins.     

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway.     

吖啶橙受溶液pH和浓度变化的光谱研究

利用荧光光谱分析仪研究吖啶橙受溶液pH和浓度变化的吸收光谱和荧光光谱的变化。实验表明,当改变吖啶橙溶液pH和浓度时,它的吸收光谱和荧光光谱发生位移。吖啶橙为1×10-6mol/L时,不同pH的吖啶橙溶液均在(490±3)nm出现一个强吸收峰,pH=6.5,吸收光谱的λm ax=430 nm,发生蓝移;而荧光光谱的λm ax随pH增大发生红移,荧光强度减弱。在浓吖啶橙溶液中,不同pH的吖啶橙溶液的吸收光谱的形状基本相同,出现两个吸收峰,λm ax1分别为(455±3)nm和(430±3)nm,λm ax2分别为(505±4)nm和(510±2)nm,吸收光谱红移;荧光光谱的λm ax均为(535±2)nm,荧光强度荧光很弱。pH相同或相近时,吖啶橙溶液的吸收光谱蓝移和荧光光谱红移,浓度越大,荧光强度越弱。还探讨了吖啶橙在水溶液中的赋存状态,结果表明,在稀溶液中,吖啶橙主要以单体的形式存在;在高浓度吖啶橙溶液中则以吖啶橙单体、二聚体,甚至多聚体形式存在。这说明溶液pH主要影响到吖啶橙分子基态的质子化和氢键的形成能力,使得分子的基态与激发态之间的能量间隔发生了变化,吖啶橙被质子化,则引起发光光谱向短波方向移动,而离解作用,则引起发光光谱向长波方向移动;吖啶橙浓度变化影响吖啶橙在水溶液赋存状态,引起吸收光谱向短波方向移动或向长波方向移动。     

The morphology and mechanical properties of phenoxy/liquid crystalline polymer blends and the effect of transesterification

Morphological, rheological, and mechanical properties of poly(hydroxy ether of bisphenol A) (phenoxy) and a Vectra liquid crystalline copolyester (LCP) blends were investigated. Scanning electron micrographs of fracture surfaces of injection-molded samples show that the LCP forms an elongated fibrous dispersion in the phenoxy matrix. As the mixing time increases, the tensile strength and modulus increase while the elongation at break remains almost constant. These improvements are attributed to the formation of LCP-grafted phenoxy by the interchange reaction between phenoxy and LCP. The interchange reaction was identified by DSC, a rheometer, and a FTIR spectrometer. The graft copolymer gives better adhesion between the two phases and thus improves mechanical properties of the blends. © 1995 John Wiley & Sons, Inc.     

Naturally functionalized triglyceride oils in interpenetrating polymer networks

The use of naturally functionalized triglyceride oils in interpenetrating polymer networks (IPNs) is reviewed. An oil bearing either hydroxyl or epoxide functionality may be crosslinked to form a soft elastomer in the presence of another monomer or network to form an IPN, or in the presence of a linear polymer, to form a semi-IPN. Polymerization and characterization of naturally functionalized triglyceride oils are mentioned, with emphasis on the distribution and effect of nontrifunctional triglycerides on elastomer properties. The simultaneous synthesis of polystyrene/functional triglyceride oil IPNs is reviewed, and several factors influencing IPN morphology and mechanical properties are discussed. The synthesis and properties of poly(ethylene terephthalate)/functional triglyceride oil semi-IPNs are emphasized, with the importance of ester interchange in the synthetic procedure, and factors influencing crystallinity and morphology are introduced.     

Hydrophilic trans‐Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling

Introduction of bioorthogonal functionalities (e.g., trans‐cyclooctene‐TCO) into a protein of interest by site‐specific genetic encoding of non‐canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine‐functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours‐long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO‐ncAAs. One derivative, DOTCO‐lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.     

Hetero‐bivalent GLP‐1/Glibenclamide for Targeting Pancreatic β‐Cells

G protein‐coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell‐specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin‐secreting pancreatic β‐cells are the sulfonylurea‐1 (SUR1) and the glucagon‐like peptide‐1 (GLP‐1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP‐1 (7–36 GLP‐1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β‐cells, by using fluorescence microscopy or time‐resolved saturation and competition binding assays, respectively. Once the ligand binds to β‐cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium‐labelled GLP‐1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high‐affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β‐cell targeting in vivo.