Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.     

以尼罗红为探针对新型两亲性树枝形聚合物微环境的研究

本工作合成了一系列外围以三缩四乙二醇单甲醚修饰的烷基芳醚骨架两亲性树枝形聚合物Gn(n=0—3),化合物通过了¹H-NMR,IR和MALDI-TOF-MS的表征.利用吸收光谱,稳态和时间分辨荧光光谱研究了水溶液中Gn对尼罗红分子的增溶作用以及Gn内部微环境的极性.研究结果表明,高代数树枝形聚合物Gn对尼罗红具有更好的增溶效果.1—3代树枝形聚合物Gn内部疏水孔腔微环境极性随代数增加而逐渐降低,G1和G2树枝形聚合物具有相似的微环境极性,而由于构象的变化使G3具有更加疏水的微环境.     

Putting a lid on protein folding: structure and function of the co-chaperonin,GroES

The co-chaperonin GroES is an essential partner in protein folding mediated by the chaperonin, GroEL. Two recent crystal structures of GroES provide a structural basis to understand how GroES forms the lid on the folding-active cis ternary complex, and how the GroEL-GroES complex enhances folding.     

Nile Red nucleoside: design of a solvatofluorochromic nucleoside as an indicator of micropolarity around DNA

The fluorophore, Nile Red, effectively works as a polarity-sensitive fluorescence probe. We have designed a new nucleoside modified by Nile Red for examining the change in the polarity of the microenvironment surrounding DNA. We synthesized a Nile Red nucleoside (1), formed by replacing nucleobases with Nile Red, through the coupling of a 2-hydroxylated Nile Red derivative and 1,2-dideoxyglycan. This nucleoside showed a high solvatofluorochromicity. The fluorescence of 1 incorporated into DNA was greatly shifted to shorter wavelength by the addition of beta-cyclodextrin. The photophysical function of the Nile Red nucleoside will be a good optical indicator for monitoring the change in the micropolarity properties at a specific site on target sequences with interaction between DNA and DNA-binding molecules.     

The mechanism of GroEL/GroES folding/refolding of protein substrates revisited

The thermodynamics and kinetics of zinc-cytochrome c (ZnCyt c) interactions with Escherichia coli molecular chaperone GroEL (Chaperonin 60; Cpn60) are described. Zinc(II)-porphyrin represents a flexible fluorescent probe for thermodynamic complex formation between GroEL and ZnCyt c, as well as for stopped-flow fluorescence kinetic experiments. Data suggests that GroEL and GroEL/GroES-assisted refolding of unfolded ZnCyt c takes place by a mechanism that is quite close to the Anfinsen Cage hypothesis for molecular chaperone activity. However, even in the presence of ATP, GroEL/GroES-assisted refolding of ZnCyt c takes place at approximately half the rate of refolding of ZnCyt c alone. On the other hand, there is little evidence for refolding behaviour consistent with the Iterative Annealing hypothesis. This includes a complete lack of GroEL or GroEL/GroES-assisted enhancement of refolding rate constant k(2) associated with the unfolding of a putative misfolded state I (Zn) on the pathway to the native state. Reviewing our data in the light of data from other laboratories, we observe that all forward rate enhancements or reductions could be accounted for in terms of thermodynamic coupling (adjusting positions of refolding equilibria) due to binding interactions between GroEL and unfolded protein substrates, driven by thermodynamic considerations. Therefore, we propose that passive kinetic partitioning should be considered the core mechanism of the GroEL/GroES molecular chaperone machinery, wherein the core function is to bind unfolded protein substrates leading to a blockade of aggregation pathways and to increases in molecular flux through productive folding pathway(s).     

Study of proteinous and micellar microenvironment using donor acceptor charge transfer fluorosensor N,N-dimethylaminonaphthyl-(acrylo)-nitrile

Interaction of charge transfer fluorophore N,N-dimethylaminonaphthyl-(acrylo)-nitrile (DMANAN) with globular proteins Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) brings forth a marked change in the position and intensity of band maxima both in case of absorption and fluorescence spectra. Spectroscopic approach has been elaborately implemented to explore the binding phenomena of the probe with HSA and BSA and it is found that the extent of binding of the probe to both serum albumins is similar in nature. Steady state fluorescence anisotropy values, fluorescence quenching study using acrylamide quencher and Red Edge Excitation Shift (REES) help in drawing reliable conclusions regarding the location of the probe molecule within the hydrophobic cavity of the proteins. An increase in fluorescence lifetime of the probe molecule solubilized in both the proteinous media also indicate that the probe is located at the motionally restricted environment inside the hydrophobic cavity of proteins and hence non-radiative channels are less operative than in the bulk water. Similarly, the variation of position and intensity of the emission maxima of DMANAN solubilized in micellar medium of Sodium Dodecyl Sulphate (SDS) also predicts well the critical micellar concentration (CMC) and polarity of micellar microenvironment.     

Characterization of local polarity and structure of Cys121 domain in beta-lactoglobulin with a new thiol-specific fluorescent probe

The design and synthesis of a new polarity-sensitive fluorescent probe, 3-(4-chloro-6-p-maleimidylphenoxyl-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine, is reported for characterizing the local polarity and structure, such as the thiol domain, of a protein. The probe comprises a polarity-sensitive fluorophore (neutral red moiety) and a thiol-specific labeling group (maleimidyl moiety). The probe exhibits a sensitive response of shift of fluorescence maximum emission wavelength to solvent polarity, but not to pH and temperature, which makes the probe suitable for determining the local polarity change of a protein denatured by pH or temperature. The application of this kind has been first demonstrated for the polarity detection of the Cys121 domain of beta-lactoglobulin. It is found that the polarity of the Cys121 domain corresponds to a dielectric constant of 17.3, and this value hardly alters after heat treatment, which may be attributed to the improved thermal reversibility by the Cys121 modification. The simple mixture of the probe and the protein at pH 5.6 is also studied, revealing that the free probe prefers to bind to an outer hydrophobic region. Heat treatment of the mixture causes the modification of Cys121 residues; this modification does not completely destroy the calyx but results in the opening of a channel for the probe to enter the calyx of beta-lactoglobulin. These results show that Cys121 plays an important role not only in the thermal reversibility but also in the accessibility of the calyx to a ligand. The strategy presented here further indicates that the combination of polarity-sensitive fluorescence probe with site-specific labeling may serve as a powerful means for elucidating structures and properties of proteins.     

A cucurbit[6]uril analogue: host properties monitored by fluorescence spectroscopy

This paper describes the host properties of a new cucurbit[6]uril analogue, studied by fluorescence and 1H NMR spectroscopy. This host has an elongated cavity with oval-shaped portals. It is intrinsically fluorescent, and more importantly, this fluorescence is sensitive to guest encapsulation, allowing for the study of the inclusion of nonfluorescent guests by fluorescence spectroscopy. In the case of benzene as guest, significant enhancement of the cucurbit[6]uril analogue host fluorescence was observed upon addition of benzene; this allowed for the determination of the binding constant for 1:1 host-guest complexation, yielding a value of K = 6900 +/- 1100 M(-1). This complexation was also studied by 1H NMR, yielding a similar value of K = 8980 +/- 500 M(-1). The binding of a much larger guest, the dye Nile Red, was also studied, but in this case using guest fluorescence. Significant suppression of the Nile Red fluorescence was observed upon 1:1 complexation with the cucurbit[6]uril analogue, with an extremely large binding constant of 8.2 +/- 0.5 x 10(6) M(-1), indicating a very strong host-guest interaction and an excellent size and shape match. In both cases, binding was much stronger than in the case of the same guests with cucurbit[6]uril itself, and in the case of Nile Red, binding was also much stronger than with modified beta- or gamma-cyclodextrins. This is partly a result of the partial aromatic nature of the host walls, which allow for pi-pi interactions not possible in cucurbiturils or cyclodextrins. The ability to study its inclusion complexes using either host or guest fluorescence, and the very high binding constants observed, illustrates the versatility and potential usefulness of this new host compound.     

Hydrogel polymer appears to mimic the performance of the GroEL/GroES molecular chaperone machine

Controlled protein folding/refolding remains a substantial challenge to the biotechnology industry. Robust and adaptable artificial polymer molecular chaperones could make important contributions towards solving this problem. Taking inspiration from the mechanism of the GroEL/GroES molecular chaperone machine, we report the preparation and testing of a selection of cross-linked thermo-responsive hydrogels, one of which is shown to assist quantitative refolding of a stringent unfolded protein substrate (mitochondrial malate dehydrogenase [mMDH]) during temperature cycling between hydrophobic and hydrophilic states. To our knowledge, this is the first hydrogel-only artificial polymer molecular chaperone to be derived, which is also potentially a generic artificial polymer molecular chaperone for use in a folding bioreactor.     

An ensemble and single-molecule fluorescence microscopy investigation of phase-separated monolayer films stained with Nile Red

Phase-separated Langmuir-Blodgett monolayer films prepared from mixtures of arachidic acid (C19H39COOH) and perfluorotetradecanoic acid (C13F27COOH) were stained via spin-casting with the polarity sensitive phenoxazine dye Nile Red, and characterized using a combination of ensemble and single-molecule fluorescence microscopy measurements. Ensemble fluorescence microscopy and spectromicroscopy showed that Nile Red preferentially associated with the hydrogenated domains of the phase-separated films, and was strongly fluorescent in these areas of the film. These measurements, in conjunction with single-molecule fluorescence imaging experiments, also indicated that a small sub-population of dye molecules localizes on the perfluorinated regions of the sample, but that this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. The relative importance of selective dye adsorption and local polarity sensitivity of Nile Red for staining applications in phase-separated LB films as well as in cellular environments is discussed in context of the experimental results.     

Probing lipid vesicles by bimolecular association and dissociation trajectories of single molecules

Vesicles prepared by DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) and SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid molecules having sizes smaller than the diffraction-limited focused laser beam have been used to confine single molecules in the laser focus. The confinement of single molecules in a volume smaller than the focused laser beam leads to a Gaussian distribution of single molecule fluorescence intensity. The interactions of single Nile Red molecules with DMPC and SOPC lipid bilayers were studied by single molecule fluorescence confocal microscopy. Nile Red molecules were observed to associate with and dissociate from individual DMPC and SOPC vesicles adsorbed on a glass surface, generating on-and-off fluctuations in a fluorescence signal representing a very low noise two-state trajectory. Off-time statistics were used to investigate the mean radius of the vesicles and the size distribution functions. The means of the on-time distributions of Nile Red in DMPC and SOPC vesicles were significantly different. The association and dissociation reactions of single Nile Red molecules with a vesicle have been studied. Features of the bimolecular interaction between the probe Nile Red and the vesicle were evaluated from the uncorrelated mean on-time and vesicle radius distributions, and the linear Nile Red concentration dependence of the mean off-time. Nile Red is shown to be a useful probe of the structural fluctuations and heterogeneity of these membrane structures, and it is a useful model with which to directly study a diffusion-influenced reversible bimolecular reaction.     

A cinnamoyl substituted Nile Red-based probe to detect hydrazine

Herein we discovered that a Nile Red-based probe with a cinnamoyl unit was highly selective and sensitive to N₂H₄. Hydrazinolysis by N₂H₄ would release a hydroxyl substituted Nile Red and result in remarkable fluorescence quench. Importantly, Cys/Hcy would not interrupt the N₂H₄ recognition. This is because, for this probe, the combination of the π-π conjugate system can stabilize the ethylene union, which results in the nucleophilic addition of the thiol group of Cys/Hcy becomes non-effective.     

Pyrene: a probe to study protein conformation and conformational changes

The review focuses on the unique spectral features of pyrene that can be utilized to investigate protein structure and conformation. Pyrene is a fluorescent probe that can be attached covalently to protein side chains, such as sulfhydryl groups. The spectral features of pyrene are exquisitely sensitive to the microenvironment of the probe: it exhibits an ensemble of monomer fluorescence emission peaks that report on the polarity of the probe microenvironment, and an additional band at longer wavelengths, the appearance of which reflects the presence of another pyrene molecule in spatial proximity (~10 ?). Its high extinction coefficient allows us to study labeled proteins in solution at physiologically relevant concentrations. The environmentally- and spatially-sensitive features of pyrene allow monitoring protein conformation, conformational changes, protein folding and unfolding, protein-protein, protein-lipid and protein-membrane interactions.     

Solubilization of hydrophobic molecules in nanoparticles formed by polymer-surfactant interactions

The interaction between the anionic surfactant, sodium dodecyl sulfate, and the polyelectrolyte, poly(diallyldimethylammonium chloride), may lead to formation of nanoparticles dispersed in water. The morphology of the resulting nanoparticles and their ability to solubilize hydrophobic molecules were evaluated. As shown by SEM and AFM imaging, the particles are spherical, having a diameter of about 20 nm. The solubilization within the nanoparticles was tested with pyrene, a fluorescence probe, and Nile Red, a solvatochromic probe. It was found that for Nile Red the solubilization within the nanoparticles is at lower polarity than for SDS micelles, and from pyrene solubilization it appears that the hydrophobicity of the nanoparticles depends on the ratio between the SDS molecules and the charge unit of the polymer.     

Probing the Polarity of Sol-Gels and Ormosils via the Absorption of Nile Red

Conventional sol-gels are rather hydrophilic. A more hydrophobic material is obtained preparing organically modified siloxanes (ormosils). The polarity-sensitive probe Nile Red (NR) was doped in various sol-gels to probe their micro-polarity. The experiments show that the NR is an excellent probe and sensitive to the polarity of its microenvironment. Spectroscopic studies reveal remarkable changes in the absorption band positions and intensities as a function of the polarity of the sol-gel, which depends on the different precursors used. Furthermore, sol aging, gelation and temporal stability as a function of different ormosils have been investigated.     

A Spectroscopic and Molecular Modelling Study of the Nature of the Association Complexes of Nile Red with Cyclodextrins

The polarity-sensitive fluorescent dye Nile Red forms association complexes with various cyclodextrins in aqueous solution. The formation of such association complexes has a significant effect on the Nile Red fluorescence, with the largest effect being observed in -cyclodextrin solution. When -cyclodextrin is used to increase the Nile Red concentration in solution, the absorption spectrum shows a large blue shift indicating the formation of an inclusion complex, but surprisingly the Nile Red fluorescence is strongly suppressed. A proposed explanation for this observation involves the formation of 1:2 host:guest complexes, in which the Nile Red guests are included as relatively non-fluorescent dimers. When the solutions were prepared by adding -cyclodextrinto a near-saturated aqueous solution of Nile Red (so that 1:1 or 2:1 complexation shouldbe favoured), significant fluorescence enhancement was observed. Analysis of thefluorescence enhancement as a function of host concentration indicated the formationof 2:1 host:guest complexes in these solutions. However, electrospray mass spectroscopic studies show no evidence for the formation of any such inclusion complexes. Furthermore, molecular modelling shows that the formation of a complex involving full insertion of Nile Red in the -cyclodextrin cavity is not stable, and will quickly eject the Nile Red guest molecule. These modelling results suggest that an association complex involving capping (via the association of Nile Red parallel to the cavity opening, or by partial insertion into the cavity) of the -cyclodextrin cavity by one or two Nile Red molecules is much more likely.     

Spectral study on the binding of gadolinium ions with apoovotransferrin

The binding of Gd3+ ion to apoovotransferrin (apoOTf) was monitored by means of UV difference spectra in 0.01M Hepes, pH 7.4 at 25 degrees C. Used 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as fluorescence probe the conformational changes of protein were studied while gadolinium ions bound to apoOTf. The results show that Gd3+ binding produces peaks at 244 and 294 nm that is the characteristic of binding at the apoOTf specific metal-binding sites. At 244 nm the molar absorptivity of Gd-apoOTf complex is (1.99+/-0.17)x10(4)cm(-1)M(-1). The apparent binding constants for the complexes of Gd3+ with apoovotransferrin are logK(1)=7.61+/-0.14 and logK(2)=4.96+/-0.26. A very large conformational change of apoovotransferrin appears when Gd3+ is bound to the N-terminal binding site. When Gd3+ is bound to C-terminal binding site there is less conformational change.     

Residue‐Specific Dynamics and Local Environmental Changes in Aβ40 Oligomer and Fibril Formation

Elucidating local dynamics of protein aggregation is crucial for understanding the mechanistic details of protein amyloidogenesis. Herein, we studied the residue‐specific dynamics and local environmental changes of Aβ40 along the course of aggregation by using para‐cyanophenylalanine (Phe_CN) as a fluorescent and vibrational probe. Our results show that the Phe_CN residues introduced at various positions all exhibited an immediate decay of fluorescence intensity, indicating a relatively synergistic process in early oligomer formation. The fast decreases in the fluorescence intensities of residues 19 and 20 in the central hydrophobic core region and residue 10 in the N‐terminal region suggest that they play crucial roles in the formation of the oligomeric core. The Phe_CN4 residue exhibits a remarkably slower decrease in fluorescence intensity, implicating its dynamic conformational characteristics in oligomer and fibril formation. Our results also suggest that the N‐terminal residues in fibrils are surrounded by a relatively hydrophobic local environment, as opposed to being solvated.     

Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer.     

Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding.