The investigation of interactions in the excited state of flavins using time-resolved spectroscopy

In paper I the results obtained with a very short (3 nsec) intense laser pulse as excitation source are described. This pulse excites such a large amount of flavin molecules into higher excited singlet and triplet states that changes in absorption of these higher excited states can be analyzed with a second less intense light flash of longer duration (5 μsec) fired synchronously with the laser pulse. The time-resolved absorption spectra obtained in this way were analyzed very carefully. When the pH was varied it was concluded that at 50 nsec after the laser pulse at least two species were present. One species could be identified as a flavin triplet. From photochemical and other data a structure for the second species was proposed, assuming the presence of flavin dimers. In such a flavin complex a very fast photoreaction occurs resulting in a photodimer, which is stable only in the excited state.In paper II another pulse technique (single photon counting) was applied to the free nucleotides FMN and FAD. These flavins were excited with a repetitive cycle of short (2 nsec) weak light flashes and the fluorescence response was measured as a function of both wavelength and time. From these time-correlated fluorescence spectra several conclusions regarding the interaction of adenine and flavin moieties (in FAD) were drawn. The most important outcome was that only the open conformation of FAD is fluorescent. An appendix (1) is added, which treats the theory and method of the time-resolved fluorescence relevant to the FAD-system.Paper III describes the results obtained with a flavoprotein (lipoamide dehydrogenase). Especially energy transfer experiments from tryptophan residues to the flavin indicate a crude distance and relative orientation between the 2 chromophores. In this study the time dependence of the tryptophan (donor) fluorescence was important. This was obtained from the phase delay of the fluorescence response on sinusoidally modulated exciting light.Paper IV gives independent evidence from the single photon counting method that the distance between a tryptophan-flavin couple is in the same order of magnitude as calculated from static analysis (paper III). Furthermore the experiments indicate that not all the tryptophans are involved in energy transfer, a result also found in paper III. The formulas describing the fluorescence decays of donor and acceptor molecules are summarized in appendix 2. A close examination of the flavin fluorescence decay demonstrates that the two FAD's occupy different sites in the dimeric protein. Several experimental tests for establishing this inequality are treated in appendix 1. The most interesting observations and significant results are discussed at the end of this thesis

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactiv

Translational and rotational motions of albumin sensed by a non-covalent associated porphyrin under physiological and acidic conditions: a fluorescence correlation spectroscopy and time resolved anisotropy study.

The interaction between a free-base, anionic water-soluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a bi-exponential decay is obtained where a short rotational correlation time (¿ int¿=¿1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin J-aggregates is induced, which can be detected by time-resolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sampl

Structural Changes of Yellow Cameleon Domains Observed by Quantitative FRET Analysis and Polarized Fluorescence Correlation Spectroscopy

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand bindin

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

An instrumental system is described for detecting and sorting single fluorescent particles such as microspheres, bacteria, viruses, or even smaller macromolecules in a flowing liquid. The system consists of microfluidic chips (biochips), computer controlled high voltage power supplies, and a fluorescence microscope with confocal optics. The confocal observation volume and detection electro-optics allow measurements of single flowing fluorescent particles. The output of the avalanche photodiode (single photon detector) is coupled to a real-time photon-burst detection device, which output can address the control of high voltage power supplies for sorting purposes. Liquid propulsion systems like electro-osmotic flow and plain electric fields to direct the particles through the observation volume have been tested and evaluated. The detection and real-time sorting of fluorescent microspheres are demonstrated. Applications of these biochips for screening of bacteriophages-type biolibraries are briefly discussed. (C) 2004 American Institute of Physics

Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes

Fluorescence of Alexa Fluor dye tracks protein folding

Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding

Effects of refractive index and viscosity on fluorescence and anisotropy decays of enhanced cyan and yellow fluorescent proteins

The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Time-resolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water-glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYF

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 apoflavodoxin's complex folding pathway