Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Concurrent presence of on- and off-pathway folding intermediates of apoflavodoxin at physiological ionic strength
Houwman, Joseline A. ; Westphal, Adrie H. ; Visser, Antonie J.W.G. ; Borst, Jan Willem ; Mierlo, Carlo P.M. van - \ 2018
Physical Chemistry Chemical Physics 20 (2018)10. - ISSN 1463-9076 - p. 7059 - 7072.

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.

Rise-Time of FRET-Acceptor Fluorescence Tracks Protein Folding
Lindhoud, S. ; Westphal, A.H. ; Mierlo, C.P.M. van; Visser, A.J.W.G. ; Borst, J.W. - \ 2014
International Journal of Molecular Sciences 15 (2014)12. - ISSN 1661-6596 - p. 23836 - 23850.
resonance energy-transfer - beta parallel protein - single-molecule fluorescence - azotobacter-vinelandii - spectroscopic ruler - refractive-index - tryptophan residue - molten globule - wild-type - pathway
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.
Phasor approaches simplify the analysis of tryptophan fluorescence data in protein denaturation studies
Bader, A.N. ; Visser, N.V. ; Amerongen, H. van; Visser, A.J.W.G. - \ 2014
Methods and Applications in Fluorescence 2 (2014)4. - ISSN 2050-6120 - 8 p.
The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods-(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy-we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process (¬GUN) 0 are obtained. The outcomes are compared to the global analysis results published previously.
Molecular dynamics simulation of energy migration between tryptophan residues in apoflavodoxin
Nunthaboot, N. ; Tanaka, F. ; Kokpol, S. ; Visser, N.V. ; Amerongen, H. van; Visser, A.J.W.G. - \ 2014
RSC Advances : An international journal to further the chemical sciences 4 (2014). - ISSN 2046-2069 - p. 31443 - 31451.
time-resolved fluorescence - azotobacter-vinelandii - anisotropy decay - force-field - flavodoxin - proteins - water - pathway - system - state
Molecular dynamics (MD) simulations over a 30 ns trajectory have been carried out on apoflavodoxin from Azotobacter vinelandii to compare with the published, experimental time-resolved fluorescence anisotropy results of F¨orster Resonance Energy Transfer (FRET) between the three tryptophan residues. MD analysis of atomic coordinates yielding both the time course of geometric parameters and the time-correlated second-order Legendre polynomial functions reflects immobilization of tryptophans in the protein matrix. However, one tryptophan residue (Trp167) undergoes flip-flop motion on the nanosecond timescale. The simulated time-resolved fluorescence anisotropy of tryptophan residues in apoflavodoxin implying a model of two unidirectional FRET pathways is in very good agreement with the experimental time-resolved fluorescence anisotropy, although the less efficient FRET pathway cannot be resolved and is hidden in the contribution of a slow protein motion.
Bioluminescent and spectroscopic properties of His-Trp-Tyr triad mutants of obelin and aequorin.
Eremeeva, E. ; Markova, S.V. ; Frank, L.A. ; Visser, A.J.W.G. ; Berkel, W.J.H. van; Vysotski, E.S. - \ 2013
Photochemistry and Photobiological Sciences 12 (2013). - ISSN 1474-905X - p. 1016 - 1024.
ca2+-regulated photoproteins - ca2+-binding photoprotein - sequence-analysis - crystal-structure - violet bioluminescence - angstrom resolution - mnemiopsis-leidyi - light-emission - w92f obelin - cloning
Ca2+-regulated photoproteins are responsible for the bioluminescence of a variety of marine organisms, mostly coelenterates. The photoproteins consist of a single polypeptide chain to which an imidazopyrazinone derivative (2-hydroperoxycoelenterazine) is tightly bound. According to photoprotein spatial structures the side chains of His175, Trp179, and Tyr190 in obelin and His169, Trp173, Tyr184 in aequorin are at distances that allow hydrogen bonding with the peroxide and carbonyl groups of the 2-hydroperoxycoelenterazine ligand. We replaced these amino acids in both photoproteins by residues with different hydrogen bond donor–acceptor capacity. All mutants exhibited luciferase-like bioluminescence activity, hardly present in the wild-type photoproteins, and showed low or no photoprotein activity, except for aeqH169Q (24% of wild-type activity), obeW179Y (23%), obeW179F (67%), obeY190F (14%), and aeqY184F (22%). The results clearly support the supposition made from photoprotein spatial structures that the hydrogen bond network formed by His–Trp–Tyr triad participates in stabilizing the 2-hydroperoxy adduct of coelenterazine. These residues are also essential for the positioning of the 2-hydroperoxycoelenterazine intermediate, light emitting reaction, and for the formation of active photoprotein. In addition, we demonstrate that although the positions of His–Trp–Tyr residues in aequorin and obelin spatial structures are almost identical the substitution effects might be noticeably different.
Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells.
Skakun, V.V. ; Engel, R. ; Borst, J.W. ; Apanasovich, V.V. ; Visser, A.J.W.G. - \ 2012
European Biophysics Journal 41 (2012)12. - ISSN 0175-7571 - p. 1055 - 1064.
photon-counting histogram - intensity distribution analysis - correlation spectroscopy - molecular brightness - protein - oligomerization - distributions - excitation - level - dimer
Fluorescence correlation spectroscopy (FCS) and photon-counting histogram (PCH) analysis use the same experimental fluorescence intensity fluctuations, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding information about the molecular brightness of fluorescent species. Analysis of both FCS and PCH results in the molecular concentration of the sample. Using a previously described global analysis procedure we report here precise, simultaneous measurements of diffusion constants and brightness values from single fluorescence fluctuation traces of green-fluorescent protein (GFP, S65T) in the cytoplasm of Dictyostelium cells. The use of a polynomial profile in PCH analysis, describing the detected three-dimensional shape of the confocal volume, enabled us to obtain well fitting results for GFP in cells. We could visualize the polynomial profile and show its deviation from a Gaussian profile.
Fluorescence of Alexa Fluor dye tracks protein folding
Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2012
PLoS ONE 7 (2012)10. - ISSN 1932-6203 - 8 p.
azotobacter-vinelandii apoflavodoxin - resonance energy-transfer - beta parallel protein - molten-globule state - flavodoxin-ii - molecules - pathway - chains - intermediate - spectroscopy
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.
Disentangling picosecond events that complicate the quantative use of the calcium sensor YC3.60
Laptenok, S. ; Stokkum, I.H.M. van; Borst, J.W. ; Oort, B.F. van; Visser, A.J.W.G. ; Amerongen, H. van - \ 2012
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 116 (2012)9. - ISSN 1520-6106 - p. 3013 - 3020.
cyan fluorescent protein - resonance energy-transfer - time-resolved spectra - living cells - fret - photoconversion - ca2+ - yfp - spectroscopy - indicators
Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.
Critical Transfer Distance Determination Between FRET Pairs
Visser, A.J.W.G. ; Vysotski, E.S. ; Lee, J. - \ 2011
Photobiological Sciences Online (2011).
Wide-Field Multi-Parameter FLIM: Long-Term Minimal Invasive Observation of Proteins in Living Cells.
Vitali, M. ; Picazo, F. ; Prokazov, Y. ; Duci, A. ; Turbin, E. ; Götze, C. ; Llopis, J. ; Hartig, R. ; Visser, A.J.W.G. ; Zuschratter, W. - \ 2011
PLoS ONE 6 (2011)2. - ISSN 1932-6203 - 12 p.
lifetime imaging microscopy - green fluorescent protein - resonance energy-transfer - single molecules - fret microscopy - global analysis - excitation - resolution - transport - oxygen
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.
Green-Fluorescent Protein from the Bioluminescent Jellyfish Clytia gregaria Is an Obligate Dimer and Does Not Form a Stable Complex with the Ca2+-Discharged Photoprotein Clytin.
Malikova, N.P. ; Visser, N.V. ; Hoek, A. van; Skakun, V.V. ; Vysotski, E.S. ; Lee, J. ; Visser, A.J.W.G. - \ 2011
Biochemistry 50 (2011)20. - ISSN 0006-2960 - p. 4232 - 4241.
vibrio-fischeri y1 - energy-transfer - correlation spectroscopy - bacterial luciferase - refractive-index - photobacterium-leiognathi - polarized fluorescence - excitation transfer - recombinant obelin - lumazine protein
Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo¨rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca2+-discharged form that is highly fluorescent (¿max = 506 nm) and its GFP (cgreGFP; ¿max = 500 nm). Ca2+-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca2+-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.
Fluorescence fluctuation analysis of receptor kinase dimerization.
Hink, M.A. ; Vries, S.C. de; Visser, A.J.W.G. - \ 2011
In: Plant Kinases: Methods and Protocols, Methods in Molecular Biology / Dissmeyer, N., Schnittger, A., Springer Science+Business Media, LLC 2011 (vol. 779 ) - ISBN 9781617792632 - 295 p.
Fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study receptor kinase mobility in planta.
Kwaaitaal, M.A.C.J. ; Schor, M. ; Hink, M.A. ; Visser, A.J.W.G. ; Vries, S.C. de - \ 2011
In: Plant Kinases: Methods and Protocols, Methods in Molecular Biology. / Dissmeyer, N., Schnittger, A., Springer - ISBN 9781617792632 - 295 p.
A general approach for detecting folding intermediates from staedy-state and time-resolved fluorescence of single-tryptophan-containing proteins
Laptenok, S. ; Visser, N.V. ; Ruchira, A. ; Westphal, A.H. ; Hoek, A. van; Mierlo, C.P.M. van; Stokkum, I.H.M. van; Amerongen, H. van; Visser, A.J.W.G. - \ 2011
Biochemistry 50 (2011)17. - ISSN 0006-2960 - p. 3441 - 3450.
azotobacter-vinelandii - apoflavodoxin - pathway - spectroscopy - peptide
During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is 4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin
Global analysis of autocorrelation functions and photon counting distributions
Skakun, V.V. ; Engel, R. ; Digris, A.V. ; Borst, J.W. ; Visser, A.J.W.G. - \ 2011
Frontiers in Bioscience (Elite Edition) 16 (2011)3. - ISSN 1945-0494 - p. 489 - 505.
In fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis the same experimental fluorescence intensity fluctuations are used, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH yielding the molecular brightness. Both FCS and PCH give information about the molecular concentration. Here we describe a global analysis protocol that simultaneously recovers relevant and common parameters in model functions of FCS and PCH from a single fluorescence fluctuation trace. The global analysis approach is described and tested with experimental fluorescence fluctuation data of enhanced green-fluorescent protein (eGFP) and dimeric eGFP (two eGFP molecules connected by a six amino acid long linker) in aqueous buffer. Brightness values and diffusion constants are recovered with good precision elucidating novel excited-state and motional properties of both proteins
Basic Photophysics.
Visser, A.J.W.G. ; Rolinski, O.J. - \ 2010
Photobiological Sciences Online (2010).
Fluorescence lifetime imaging microscopy in life sciences
Borst, J.W. ; Visser, A.J.W.G. - \ 2010
Measurement Science and Technology 21 (2010)10. - ISSN 0957-0233 - 21 p.
resonance energy-transfer - protein-protein interactions - quantitative fret analysis - living plant-cells - correlation spectroscopy - live cells - global analysis - streak camera - modulation fluorometry - polarized fluorescence
Fluorescence lifetime imaging microscopy (FLIM) and fluorescence anisotropy imaging microscopy (FAIM) are versatile tools for the investigation of the molecular environment of fluorophores in living cells. Owing to nanometre-scale interactions via Förster resonance energy transfer (FRET), FLIM and FAIM are powerful microscopy methods for the detection of conformational changes and protein–protein interactions reflecting the biochemical status of live cells. This review provides an overview of recent advances in photonics techniques, quantitative data analysis methods and applications in the life sciences
Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence
Laptenok, S. ; Borst, J.W. ; Mullen, K.M. ; Stokkum, I.H.M. van; Visser, A.J.W.G. ; Amerongen, H. van - \ 2010
Physical Chemistry Chemical Physics 12 (2010)27. - ISSN 1463-9076 - p. 7593 - 7602.
nonlinear least-squares - quantitative fret analysis - resolved spectra - correlation spectroscopy - picosecond fluorescence - reference convolution - refractive-index - proteins - excitation - precision
A methodology is described for the quantitative determination of Förster resonance energy transfer (FRET) in live cells using the rise time of acceptor fluorescence as determined with fluorescence lifetime imaging microscopy (FLIM). An advantage of this method is that only those molecules that are involved in the energy-transfer process are monitored. This contrasts with current methods that measure either steady-state fluorescence of donor and acceptor molecules or time-resolved fluorescence of donor molecules, and thereby probe a mixture of donor molecules that are involved in FRET and those that are fluorescent but not involved in FRET. The absence of FRET can, for instance, be due to unwanted acceptor bleaching or incomplete maturing of visible proteins that should act as acceptor molecules. In addition, parameters describing the rise of acceptor fluorescence and the decay of donor fluorescence can be determined via simultaneous global analysis of multiple FLIM images, thereby increasing the reliability of the analysis. In the present study, plant protoplasts transfected with fusions of visible fluorescent proteins are used to illustrate the new data analysis method. It is demonstrated that the distances estimated with the present method are substantially smaller than those estimated from the average donor lifetimes, due to a fraction of non-transferring donor molecules. Software to reproduce the presented results is provided in an open-source and freely available package called "TIMP" for "The R project for Statistical Computing"
Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs
Visser, A.J.W.G. ; Laptenok, S. ; Visser, N.V. ; Hoek, A. van; Birch, D.J.S. ; Brochon, J.C. ; Borst, J.W. - \ 2010
European Biophysics Journal 39 (2010)2. - ISSN 0175-7571 - p. 241 - 253.
lifetime imaging microscopy - maximum-entropy method - energy-transfer - living cells - polarized fluorescence - flavin fluorescence - lipoamide dehydrogenase - glutathione-reductase - dynamics - photoconversion
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-inactive
The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding.
Eremeeva, E. ; Markova, S.V. ; Westphal, A.H. ; Visser, A.J.W.G. ; Berkel, W.J.H. van; Vysotski, S. - \ 2009
FEBS Letters 583 (2009)12. - ISSN 0014-5793 - p. 1939 - 1944.
crystal-structure - ca2+-regulated photoproteins - violet bioluminescence - angstrom resolution - recombinant obelin - w92f obelin - calcium - regeneration - apoaequorin - expression
The intrinsic fluorescence of two apo-photoproteins has been characterized and its concentration-dependent quenching by coelenterazine has been for the first time applied to determine the apparent dissociation constants for coelenterazine binding with apo-aequorin (1.2±0.12µM) and apo-obelin (0.2±0.04µM). Stopped-flow measurements of fluorescence quenching showed that coelenterazine binding is a millisecond-scale process, in contrast to the formation of an active photoprotein complex taking several hours. This finding evidently shows that the rate-limiting step of active photoprotein formation is the conversion of coelenterazine into its 2-hydroperoxy derivative
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