RESUMO
Optical tissue clearing refers to physico-chemical treatments which make thick biological samples transparent by removal of refractive index gradients and light absorbing substances. Although tissue clearing was first reported in 1914, it was not widely used in light microscopy until 21th century, because instrumentation of that time did not permit to acquire and handle images of thick (mm to cm) samples as whole. Rapid progress in optical instrumentation, computers and software over the last decades made micrograph acquisition of centimeter-thick samples feasible. This boosted tissue clearing use and development. Numerous diverse protocols have been developed. They use organic solvents or water-miscible substances, such as detergents and chaotropic agents; some protocols require application of electric field or perfusion with special devices. There is no 'best-for-all' tissue clearing method. Depending on the case, one or another protocol is more suitable. Most of protocols require days or even weeks to complete, thus choosing an unsuitable protocol may cause an important waste of time. Several inter-dependent parameters should be taken into account to choose a tissue clearing protocol, such as: (1) required image quality (resolution, contrast, signal to noise ratio etc), (2) nature and size of the sample, (3) type of labels, (4) characteristics of the available instrumentation, (5) budget, (6) time budget, and (7) feasibility. Present review focusses on the practical aspects of various tissue clearing techniques. It is aimed to help non-experts to choose tissue clearing techniques which are optimal for their particular cases.
Assuntos
Técnicas de Preparação Histocitológica/métodos , Animais , Humanos , Microscopia/métodosRESUMO
Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid's position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.
Assuntos
Microscopia de Fluorescência/métodos , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , HumanosRESUMO
Conventional fragments of fluorescent proteins used in bimolecular fluorescence complementation technique (BiFC), form light-emitting species only when they are kept in close proximity by interacting proteins of interest. By contrast, certain fluorescent protein fragments complement spontaneously, namely those corresponding to the 1st to 10th beta-strands (GFP1-10) and the 11th beta-strand of superfolder GFP (GFP11). They were designed as folding reporters for high throughput expression and structure biology. Besides, for light microscopy, self-associating fluorescent protein fragments constitute a valuable and sometimes unique tool. The GFP11 tag is very advantageous when a full-length fluorescent protein cannot be fused to a protein of interest, namely for live imaging of certain pathogens. Self-associating GFP fragments enable live labelling of specific synapses, visualization of proteins topology and their exposure to particular subcellular compartments. Present review aims to attract attention of scientific community to these tools and to inspire their further development and applications.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Sondas Moleculares/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Sondas Moleculares/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Ligação Proteica , Multimerização ProteicaRESUMO
Mitochondrial DNA molecules coated with proteins form compact particles called mitochondrial nucleoids. They are redistributed within mitochondrial network undergoing morphological changes. The straightforward technique to characterize nucleoids' motions is fluorescence microscopy. Mitochondrial nucleoids are commonly labelled with fluorescent protein tags, which is not always feasible and was reported to cause artifacts. Organic DNA-binding dyes are free of these drawbacks, but they lack specificity to mitochondrial DNA. Here, considering physico-chemical properties of such dyes, we achieved preferential live-cell labelling of mitochondrial nucleoids by a nucleic acid staining dye SYBR Gold. It enabled time-lapse imaging of mitochondrial nucleoids by structured illumination microscopy and quantification of their motions.
Assuntos
Corantes/metabolismo , DNA Mitocondrial/ultraestrutura , Mitocôndrias/ultraestrutura , Compostos Orgânicos/metabolismo , Células A549 , Animais , Chlorocebus aethiops , DNA Mitocondrial/metabolismo , Células HeLa , Humanos , Microscopia/métodos , Mitocôndrias/metabolismo , Imagem com Lapso de Tempo , Células VeroRESUMO
Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a "back-to-back" fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.
Assuntos
Imunoglobulina G/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Dimerização , Humanos , Modelos Moleculares , Transdução de Sinais/fisiologia , Proteínas RoundaboutRESUMO
Dynamic studies of influenza virus infection in the live cells are limited because of the lack of appropriate methods for non-invasive detection of the viral components. Using the split-GFP strategy, we have recently developed and characterized an unimpaired recombinant influenza A virus encoding a tagged PB2 subunit of RNA-dependent RNA polymerase, which enabled continuous real-time visualization of the viral ribonucleoproteins (vRNPs) in living cells (Avilov, Moisy, Munier, Schraidt, Naffakh and Cusack [12]). Here, using this virus, we studied vRNP trafficking and interaction with Rab11 in the context of quasi-wild type infection. In agreement with recent reports, we observed that upon nuclear export, progeny vRNPs accumulate in the particles containing Rab11, a multifunctional protein involved in vesicle trafficking which resides at recycling endosomes. Fluorescence resonance energy transfer microscopy indicated a distance <10nm between PB2 and Rab11, suggesting that a direct interaction occurs. Single particle tracking analysis showed that most of the motions of vRNP-positive particles in infected cells are slow, while rapid directional motions intermittently occur. Analysis focused on these intermittent motions indicated that depolymerization of either microtubules or actin filaments moderately reduced their occurrence, while disruption of both cytoskeleton components in combination suppressed the rapid motions entirely. Thus, the split-GFP based virus enabled us to obtain a live-cell based confirmation for the model of vRNP trafficking which assumes accumulation of vRNP in recycling endosomes through a direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.
Assuntos
Endossomos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Vírus da Influenza A/fisiologia , Nucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Linhagem Celular , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/genética , Humanos , Microscopia de Fluorescência , Mapeamento de Interação de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coloração e Rotulagem/métodos , Proteínas Virais/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.
Assuntos
Proteína HMGB1/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Sequência de Aminoácidos , Linhagem Celular , Proteína HMGB1/genética , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Influenza Humana/virologia , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Proteínas do Core Viral/química , Proteínas do Core Viral/genéticaRESUMO
Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Sítios de Ligação , Linhagem Celular Transformada , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Células HEK293 , HIV-1/metabolismo , Humanos , Modelos Moleculares , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
Since currently available therapies against HIV/AIDS still show important drawbacks, the development of novel anti-HIV treatments is a key issue. We recently characterized methylated oligoribonucleotides (mONs) that extensively inhibit HIV-1 replication in primary T cells at nanomolar concentrations. The mONs were shown to target both HIV-1 reverse transcriptase (RT) and the nucleocapsid protein (NC), which is an essential partner of RT during viral DNA synthesis. To further understand the mechanism of such mONs, we studied by isothermal titration calorimetry and fluorescence-based techniques their NC binding properties and ability to inhibit the nucleic acid chaperone properties of NC. Notably, we investigated the ability of mONs to inhibit the NC-induced destabilization of the HIV-1 cTAR (complementary DNA sequence to TAR [transactivation response element]) stem-loop and the NC-promoted cTAR annealing to its complementary sequence, required at the early stage of HIV-1 viral DNA synthesis. Moreover, we compared the activity of the mONs to that of a number of modified and nonmodified oligonucleotides. Results show that the mONs inhibit NC by a competitive mechanism whereby the mONs tightly bind the NC peptide, mainly through nonelectrostatic interactions with the hydrophobic platform at the top of the NC zinc fingers. Taken together, these results favor the notion that the mONs impair the process of the RT-directed viral DNA synthesis by sequestering NC molecules, thus preventing the chaperoning of viral DNA synthesis by NC. These findings contribute to the understanding of the molecular basis for NC inhibition by mONs, which could be used for the rational design of antiretroviral compounds targeting HIV-1 NC protein.
Assuntos
HIV-1/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Oligorribonucleotídeos/farmacologia , Sequência de Aminoácidos , Sequência de Bases , DNA Viral/biossíntese , Transcriptase Reversa do HIV/antagonistas & inibidores , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismoRESUMO
Studies on the intracellular trafficking of influenza virus ribonucleoproteins are currently limited by the lack of a method enabling their visualization during infection in single cells. This is largely due to the difficulty of encoding fluorescent fusion proteins within the viral genome. To circumvent this limitation, we used the split-green fluorescent protein (split-GFP) system (S. Cabantous, T. C. Terwilliger, and G. S. Waldo, Nat. Biotechnol. 23:102-107, 2005) to produce a quasi-wild-type recombinant A/WSN/33/influenza virus which allows expression of individually fluorescent PB2 polymerase subunits in infected cells. The viral PB2 proteins were fused to the 16 C-terminal amino acids of the GFP, whereas the large transcomplementing GFP fragment was supplied by transient or stable expression in cultured cells that were permissive to infection. This system was used to characterize the intranuclear dynamics of PB2 by fluorescence correlation spectroscopy and to visualize the trafficking of viral ribonucleoproteins (vRNPs) by dynamic light microscopy in live infected cells. Following nuclear export, vRNPs showed a transient pericentriolar accumulation and intermittent rapid (â¼1 µm/s), directional movements in the cytoplasm, dependent on both microtubules and actin filaments. Our data establish the potential of split-GFP-based recombinant viruses for the tracking of viral proteins during a quasi-wild-type infection. This new virus, or adaptations of it, will be of use in elucidating many aspects of influenza virus host cell interactions as well as in screening for new antiviral compounds. Furthermore, the existence of cell lines stably expressing the complementing GFP fragment will facilitate applications to many other viral and nonviral systems.
Assuntos
Proteínas de Fluorescência Verde/genética , Vírus da Influenza A/fisiologia , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Replicação Viral , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Vírus da Influenza A/genéticaRESUMO
Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly of great significance in understanding the emergence of new strains with pandemic potential. In this study, we systematically investigated the interactions between the polymerase subunits and their localization in living cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2. Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together, our results demonstrate which of the previously discussed influenza virus polymerase transport models operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects of different host range mutations in the future.
Assuntos
Núcleo Celular/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Espectrometria de Fluorescência/métodos , Linhagem Celular , Humanos , Frações Subcelulares/enzimologiaRESUMO
The nucleocapsid protein (NC) of HIV-1 is a highly conserved protein essential for the virus life cycle that constitutes an attractive target for new antiviral agents. Most NC functions rely on its binding to the HIV-1 genomic RNA and its DNA copies that contain multiple and possibly interdependent binding sites. Therefore, a detailed understanding of NC binding requires a site-specific experimental approach. We have recently shown that 2-aminopurine (2Ap), a fluorescent adenine analogue, can site-selectively probe the binding of NC. Here, we introduced 2Ap at various positions of model single-stranded dodecanucleotides containing two TG motifs which constitute putative specific binding sites. Steady-state and time-resolved fluorescence experiments indicated that NC binding strongly increased the fluorescence quantum yield of 2AP by reducing the dynamic quenching of 2Ap by its close neighbors and slowing the picosecond to nanosecond conformational fluctuations of the oligonucleotides. The dodecanucleotides were found to bind two NC molecules at physiological salt concentrations, confirming the preferential binding of NC to TG motifs and an occluded binding site size for NC of five to six bases. Using the NC-induced changes in 2Ap fluorescence, we determined the microscopic affinity constants of the individual binding sites and showed that affinities can significantly differ from one site to another within the same dodecanucleotide, depending on the position of the TG dinucleotide and the nature of its close neighbors. Moreover, our data suggest that binding of NC even to close binding sites shows no strong cooperativity.
Assuntos
Oligonucleotídeos/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , 2-Aminopurina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , HIV-1/química , Cinética , Dados de Sequência Molecular , Ligação ProteicaRESUMO
The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.
Assuntos
Cromonas/farmacologia , Corantes Fluorescentes/farmacologia , alfa-Cristalinas/química , Animais , Fenômenos Biofísicos , Biofísica , Bovinos , Cromonas/química , Cisteína/química , Ligação de Hidrogênio , Cristalino/metabolismo , Modelos Químicos , Estrutura Quaternária de Proteína , Prótons , Espectrometria de Fluorescência , Espectrofotometria , Temperatura , Fatores de TempoRESUMO
Ratiometric fluorescent probes based on 3-hydroxyflavone (3HF) are highly sensitive tools for studying polarity, hydration, electronic polarizability, and electrostatics in different microheterogeneous systems, including protein molecules. In the present work, a reactive derivative of 3HF, 6-bromomethyl-4'-diethylamino-3-hydroxyflavone, recently synthesized in our group, was applied to label covalently bovine lens alpha-crystallin. The labeling of SH and NH(2) groups are clearly distinguished by spectroscopic criteria. We observe that the NH(2) labeling creates the positive charge in the proximity to fluorophore, which results in strong internal Stark effect producing the shift in excitation spectrum by ca. 15 nm. Analysis of excitation-dependent fluorescence spectra allows separation of the emission profiles of these SH- and NH(2)-labeled species. Applying recently developed multiparametric analysis of the obtained emission spectra, we described the physicochemical properties of the sites of SH and NH(2) labeling in alpha-crystallin. The site of SH labeling has medium-low polarity (dielectric constant, epsilon = 4.9 +/- 0.9) is protic, and does not contain proximal aromatic residues (according to the obtained refractive index, n = 1.41 +/- 0.14). The site of NH(2) labeling is also of medium-low polarity. The novel label due to its two-wavelength ratiometric response and high sensitivity to the type of labeling may offer new possibilities in the studies of structure, dynamics, and interactions of proteins by probing their SH- and NH(2)-labeling sites.
Assuntos
Cisteína/química , Flavonoides/química , Corantes Fluorescentes/química , Lisina/química , Coloração e Rotulagem/métodos , alfa-Cristalinas/química , Estrutura Molecular , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.
Assuntos
Proteínas/química , Proteínas/metabolismo , Ressonância de Plasmônio de Superfície , alfa-Cristalinas/química , alfa-Cristalinas/metabolismo , Animais , Bovinos , Ditiotreitol/farmacologia , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Análise EspectralRESUMO
alpha-Crystallin, an oligomeric protein in vertebrate eye lens, is a member of the small heat-shock protein family. Several papers pointed out that its chaperone-like activity could be enhanced by increasing the temperature. We demonstrate in the present study that structural perturbations by high hydrostatic pressures up to 300 MPa also enhance this activity. In contrast with temperature-induced changes, the pressure-induced enhancement is reversible. After pressure release, the extra activity is lost with a relaxation time of 2.0+/-0.5 h. Structural alterations contributing to the higher activity were studied with IR and fluorescence spectroscopy, and light-scattering measurements. The results suggest that while the secondary structure barely changes under pressure, the interactions between the subunits weaken, the oligomers dissociate, the area of accessible hydrophobic surfaces significantly increases and the environment of tryptophan residues becomes slightly more polar. It seems that structural flexibility and the total surface area of the oligomers are the key factors in the chaperone capacity, and that the increase in the chaperone activity does not require the increase in the oligomer size as was assumed previously [Burgio, Kim, Dow and Koretz (2000) Biochem. Biophys. Res. Commun. 268, 426-432]. After pressure release, the structure of subunits are reorganized relatively quickly, whereas the oligomer size reaches its original value slowly with a relaxation time of 33+/-4 h. In our interpretation, both the fast and slow structural rearrangements have an impact on the functional relaxation.