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Johan Hofkens

Publications and source records attributed to Johan Hofkens.

At least 37 records · Page 2Linked to original sources
PubMed2005

Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed.

Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.

Decarboxylation↗
PubMed2005

Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa.

Reversible photoswitching of individual molecules has been demonstrated for a number of mutants of the green fluorescent protein (GFP). To date, however, a limited number of switching events with slow response to light have been achieved at the single-molecule level. Here, we report reversible photoswitching characteristics observed in individual molecules of Dronpa, a mutant of a GFP-like fluorescent protein that was cloned from a coral Pectiniidae. Ensemble spectroscopy shows that intense irradiation at 488 nm changes Dronpa to a dim protonated form, but even weak irradiation at 405 nm restores it to the bright deprotonated form. Although Dronpa exists in an acid-base equilibrium, only the photoinduced protonated form shows the switching behavior. At the single-molecule level, 488- and 405-nm lights can be used to drive the molecule back and forth between the bright and dim states. Such reversible photoswitching could be repeated >100 times. The response speed to irradiation depends almost linearly on the irradiation power, with the response time being in the order of milliseconds. The perfect reversibility of the Dronpa photoswitching allows us to propose a detailed model, which quantitatively describes interconversion among the various states. The fast response of Dronpa to light holds great promise for following fast diffusion or transport of signaling molecules in live cells.

Animals↗
PubMed2005

Stretched exponential decay and correlations in the catalytic activity of fluctuating single lipase molecules.

Single-molecule techniques offer a unique tool for studying the dynamical behavior of individual molecules and provide the possibility to construct distributions from individual events rather than from a signal stemming from an ensemble of molecules. In biological systems, known for their complexity, these techniques make it possible to gain insights into the detailed spectrum of molecular conformational changes and activities. Here, we report on the direct observation of a single lipase-catalyzed reaction for extended periods of time (hours), by using confocal fluorescence microscopy. When adding a profluorescent substrate, the monitored enzymatic activity appears as a trajectory of "on-state" and "off-state" events. The waiting time probability density function of the off state and the state-correlation function fit stretched exponentials, independent of the substrate concentration in a certain range. The data analysis unravels oscillations in the logarithmic derivative of the off-state waiting time probability density function and correlations between off-state events. These findings imply that the fluctuating enzyme model, which involves a spectrum of enzymatic conformations that interconvert on the time scale of the catalytic activity, best describes the observed enzymatic activity. Based on this model, values for the coupling and reaction rates are extracted.

Biophysical Phenomena↗
PubMed2005

Electron transfer at the single-molecule level in a triphenylamine-perylene imide molecule.

Photoinduced electron transfer (ET) processes in a donor-acceptor system based on triphenylamine and perylene imide have been studied at the single-molecule (SM) and ensemble levels. The system exists as two isomers, one of which undergoes forward and reverse ET in toluene with decay constants of 3.0 and 2.2x10(9) s(-1), respectively, resulting in the dual emission of quenched and delayed fluorescence while the other isomer remains ET-inactive. The fluorescence of both isomers is heavily quenched in the more polar solvent, diethyl ether, by ET. A broad range of ET dynamics is seen at the SM level in polystryene with the two isomers nonresolvable indicating that the local nanoenvironment of the SMs varies considerably throughout the polymer matrix. Both the electronic coupling and the driving force for ET are shown to influence the ET dynamics. Many fluorescence trajectories of SMs show long periods (tens of milliseconds to seconds) where the count rate is attenuated either partly (a "dim" state) or to the background level (an "off-time"). During these periods, the reduction or interruption of emission is attributed to cycles of rapid charge separation followed by charge recombination to the ground state reducing the fluorescence quantum yield of the SM.

Journal Article↗
PubMed2004

Fluorescence lifetimes and emission patterns probe the 3D orientation of the emitting chromophore in a multichromophoric system.

In this Communication, we report on the fluorescence behavior of an individual first generation multichromophoric dendrimer. The fact that each of the chromophores in time acts as a fluorescent trap is demonstrated by directly probing the dipole orientation of the emitting chromophore by means of defocused wide-field imaging and comparing experimental and calculated emission patterns. It is shown that in such cases the electromagnetic boundary condition effect results in discrete changes in the fluorescence lifetime as a function of time for individual dendrimers.

Journal Article↗
PubMed2004

Probing conformational dynamics in single donor-acceptor synthetic molecules by means of photoinduced reversible electron transfer.

We use single-molecule fluorescence lifetimes to probe dynamics of photoinduced reversible electron transfer occurring between triphenylamine (donor) and perylenediimide (acceptor) in single molecules of a polyphenylenic rigid dendrimer embedded in polystyrene. Here, reversible electron transfer in individual donor-acceptor molecules results in delayed fluorescence that is emitted with a high photon count rate. By monitoring fluorescence decay times on a photon-by-photon basis, we find fluctuations in both forward and reverse electron transfer spanning a broad time range, from milliseconds to seconds. Fluctuations are induced by conformational changes in the dendrimer structure as well by polystyrene chain reorientation. The conformational changes are related to changes in the dihedral angle of adjacent phenyl rings located in the dendritic branch near the donor transferring the charge, a torsional motion that results in millisecond fluctuations in the "through-bond" donor-acceptor electronic coupling. Polymer chain reorientation leads to changes in the local polarity experienced by the donors and to changes in the solvation of the charge-separated state. As a result, switching between different donor moieties within the same single molecule becomes possible and induces fluctuations in decay time on a time scale of seconds.

Journal Article↗
PubMed2004

Single-molecule conformations probe free volume in polymers.

In this communication, we showed that the conformational dynamics of the tetraphenoxy-perylenetetracarboxy diimide single dye molecule embedded in different polymer matrixes allows a direct visualization of the local free volume. By monitoring the fluorescence lifetime of the dye molecule in time and at different locations in the matrix, different lifetimes were observed. Attributed to two distinct stable conformations of the dye molecule, the lifetime variations permitted characterization of the spatial distribution and temporal dynamics of free volume.

Journal Article↗
PubMed2004

Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin.

We report on single-molecule fluorescence measurements performed on the phycobiliprotein allophycocyanin (APC). Our data support the presence of a unidirectional Förster-type energy transfer process involving spectrally different chromophores, alpha84 (donor) and beta84 (acceptor), as well as of energy hopping amongst beta84 chromophores. Single-molecule fluorescence spectra recorded from individual immobilized APC proteins indicate the presence of a red-emitting chromophore with emission peaking at 660 nm, which we connect with beta84, and a species with the emission peak blue shifted at 630 nm, which we attribute to alpha84. Polarization data from single APC trimers point to the presence of three consecutive red emitters, suggesting energy hopping amongst beta84 chromophores. Based on the single-molecule fluorescence spectra and assuming that emission at the ensemble level in solution comes mainly from the acceptor chromophore, we were able to resolve the individual absorption and emission spectra of the alpha84 and beta84 chromophores in APC.

Dose-Response Relationship, Radiation↗
PubMed2003

Intramolecular directional förster resonance energy transfer at the single-molecule level in a dendritic system.

We report on the directional Förster resonance energy transfer (FRET) process taking place in single molecules of a first (T1P4) and a second (T2P8) generation of a perylenemonoimide (P)-terrylenediimide (T)-based dendrimer in which the chromophores are separated by rigid polyphenylene arms. At low excitation powers, single-molecule detection and spectroscopy of T1P4 and T2P8 dendrimers point to a highly efficient directional FRET from P donors to the central T acceptor, optical excitation at 488 nm resulting in exclusively acceptor emission in the beginning of the detected fluorescence intensity. Donor emission is seen only upon the bleaching of the acceptor. High-resolution time-resolved single-molecule fluorescence data measured with a microchannel plate photomultiplier reveal, for T2P8, a broad range of FRET rates as a result of a broad range of distances and orientations experienced by the donor-acceptor dendrimers when immobilized in a polymer matrix. Single-molecule data from T2P8 on 488 nm excitation are indicative for the presence, after terrylenediimide bleaching, of a P-P excited dimer characterized by a broad emission spectrum peaking around 600 nm and by fluctuating fluorescence decay times. At high excitation powers, single T1P4 and T2P8 molecules display simultaneous emission from both donor and acceptor chromophores. The effect, called "exciton blockade", occurs due to the presence of multiple excitations in a single molecule.

Journal Article↗
PubMed2003

Revealing competitive Forster-type resonance energy-transfer pathways in single bichromophoric molecules.

We demonstrate measurements of the efficiency of competing Förster-type energy-transfer pathways in single bichromophoric systems by monitoring simultaneously the fluorescence intensity, fluorescence lifetime, and the number of independent emitters with time. Peryleneimide end-capped fluorene trimers, hexamers, and polymers with interchromophore distances of 3.4, 5.9, and on average 42 nm, respectively, served as bichromophoric systems. Because of different energy-transfer efficiencies, variations in the interchromophore distance enable the switching between homo-energy transfer (energy hopping), singlet-singlet annihilation, and singlet-triplet annihilation. The data suggest that similar energy-transfer pathways have to be considered in the analysis of single-molecule trajectories of donor/acceptor pairs as well as in natural and synthetic multichromophoric systems such as light-harvesting antennas, oligomeric fluorescent proteins, and dendrimers. Here we report selectively visualization of different energy-transfer pathways taking place between identical fluorophores in individual bichromophoric molecules.

Fluorescence Resonance Energy Transfer↗
PubMed2003

Single-molecule surface enhanced resonance Raman spectroscopy of the enhanced green fluorescent protein.

In the present contribution, we demonstrated that surface-enhanced resonance Raman scattering spectra from single green fluorescent proteins (GFPs) were obtained. The most important findings are the direct detection of the conversion between a deprotonated and a protonated form of the chromophore at the single-molecule level via the corresponding vibrational fingerprints, and the fact that the enhanced green fluorescent protein (EGFP) also shows a high surface enhanced resonance Raman scattering (SERRS) signal. Our findings show the potential of the technique to study structural dynamics of protein molecules at a single-molecule level.

Green Fluorescent Proteins↗
PubMed2003

Photoluminescence intensity fluctuations and electric-field-induced photoluminescence quenching in individual nanoclusters of poly(phenylenevinylene).

Substantial fluctuations of the fluorescence intensity have been detected for single clusters of poly(phenylenevinylene) containing more than 75 polymer chains or 30,000 monomer units. To the best of our knowledge, this is the first time such fluctuations (which resemble the "blinking" effect in single-molecule fluorescence) have been reported for such a large molecular ensemble containing several macromolecules. Together with the distinct jumps, smooth fluctuations of the fluorescence intensity, with characteristic times from milliseconds to seconds, were observed. This fact distinguishes the fluorescence behaviour of the polymer clusters from that of other multichromophoric systems such as the single chains of conjugated polymers reported in the literature. The consecutive or simultaneous switching of one or several emitting sites from the "on" to "off" state does not explain the character of the fluctuations observed. We suggest that the quenching of the light-emitting exciton by a long-lived species, such as, for example, polarons, plays an important role in these unusual fluctuations. Electric field induced fluorescence quenching differs significantly for different clusters. It is proposed that this fluorescence was mainly quenched by polarons injected from the electrodes in the presence of an electric field. The specific behaviour of each cluster is explained by suggesting a different position of the clusters with respect to the electrodes.

Journal Article↗
PubMed2002

Excitation energy transfer in dendritic host-guest donor-acceptor systems.

We report on a study of a physically formed host-guest system, which was designed to be investigated by fluorescence energy transfer. All donor and acceptor molecules used were cyanine dyes. Investigation was performed at the ensemble level as well as at the single-molecule level. The ensemble measurements revealed a distribution of binding sites as well for the donor as for the acceptor. Accordingly, we found a distribution of the energy transfer efficiency. At the single-molecule level, these distributions are still present. We could discriminate entities that show very efficient energy transfer, some that do not show any energy transfer and systems whose energy transfer efficiency is only about 50%. The latter allowed the time-resolved detection of energy transfer of single entities through the acceptor decay. Finally, we discuss the observation that the energy transfer efficiency fluctuates as a function of time.

Journal Article↗
PubMed2002

Antibunching in the emission of a single tetrachromophoric dendritic system.

The photophysics of a dendrimer containing four chromophores are investigated at the single-molecule level. First, the multichromophoric character of single dendrimers' absorption is probed by modulating the linear polarization of the excitation beam. Subsequently, using circular polarization, the same dendrimers are excited, and their fluorescence transients are recorded. Using pulsed excitation in combination with the classical Hanbury-Brown and Twiss coincidence setup the presented data demonstrate that efficient singlet-singlet annihilation ensures that always only one photon is emitted even when several excitations are generated in an individual multichromophoric molecule.

Journal Article↗