Fluorescence resonance energy transfer studies on the proximity between lysine-107 and cysteine-239 in rabbit muscle aldolase

Spatial relationships between Lys-107, which binds the C-6 phosphate group of the substrate, and fast-reacting Cys-239, located outside the active site of rabbit muscle aldolase, were studied by means of resonance energy transfer. The Lys-107 residue was covalently linked to pyridoxal phosphate (fluorescence donor) and the Cys-239 residue was modified by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (fluorescence acceptor). The energy transfer between donor and acceptor has been demonstrated. The steady-state and the lifetime measurements indicate that in solution the distance between Lys-107 and Cys-239 in the aldolase molecule is 12.4 A assuming chi 2 = 2/3.     

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau相似文献   

Studies on Chromophore Coupling in Isolated Phycobiliproteins: II. Picosecond Energy Transfer Kinetics and Time-Resolved Fluorescence Spectra of C-Phycocyanin from Synechococcus 6301 as a Function of the Aggregation State

The fluorescence kinetics of C-Phycocyanin in the monomeric, trimeric, and hexameric aggregation states has been measured as a function of the emission wavelength with picosecond resolution using the single-photon timing technique. All the decay curves measured at the various emission wavelengths were analyzed simultaneously by a global data analysis procedure. A sum of four exponentials was required to fit the data for the monomers and trimers. Only in the case of the hexamers, a three-exponential model function proved to be nearly sufficient to describe the experimental decays. The lifetime of those fluorescence components reflecting energy transfer decreased with increasing aggregation. This is due to the increased number of efficient acceptor molecules next to a donor in the higher aggregates. In all aggregates the shortest-lived component, ranging from 50 ps for monomer to 10 ps for hexamers, is observed as a decay term (positive amplitude) at short emission wavelength. At long emission wavelength it turns into a rise term (negative amplitude). The lifetime of a second ps-component ranges from 200 ps for monomers to 50 ps for hexamers. The long-lived (ns) fluorescence is inhomogeneous in monomers and trimers, showing two lifetimes of ~0.6 and 1.3 ns. The latter one carries the larger amplitude. The amplitudes of the kinetic components in the fluorescence decays are presented as time-resolved component spectra. A theoretical model has been derived to rationalize the observed fluorescence kinetics. Using symmetry arguments, it is shown that the fluorescence kinetics of C-Phycocyanin is expected to be characterized by three exponential kinetic components, independent of the aggregation state. An analytical expression is derived, which allows us to gain a detailed understanding of the origin of the different kinetic components and their associated time-resolved spectra. Numerical calculations of time-resolved spectra are compared with the experimental data.     

Cobalt probing of structural alternatives for insulin in solution

Inorganic anions and phenolic compounds make the subunits of insulin hexamers undergo the T----R transition whereby the extended N-terminal B chain becomes helical and the octahedral metal coordination tetrahedral. The role of the metal ions is permissive. With cresol the transition is also undergone by metal-free hexamers. For coordinative reasons only zinc insulin can be transformed by moderate concentrations of inorganic anions. At higher concentrations and particularly with cresol transformation is also possible if Zn2+ is replaced by other metal ions. Owing to its d--d transitions in the visible cobalt lends itself as a spectroscopic probe for studying the interdependence of transformation and coordination. The transformation-related change in coordination is reflected in both the isotropic absorption and the CD spectrum. Cresol achieves T6----R6 transformation whereas that induced by SCN- ions is T6----T'3R3 with only the axial metal-binding site being realized in the R3 trimer. The spectral effects of the transformation of the two trimers are not additive; an extra contribution seems to be indicative of trimer/trimer interaction. Oxidation of 2 Co2+ insulin to a certain extent affects the structure of insulin; a characteristic positive band appears at 251 nm. Because of its extremely stable and exclusively octahedral complexes the Co3+ ion most strongly withstands transformation. The oxidation of tetrahedrally liganded Co2+ ions in R3 trimers proceeds with reduced velocity. Independent transformation of the Zn2+ trimers is possible in Zn2+/Co3+ metal hybrids of insulin.     

Highly sensitive detection of hybridization of oligonucleotides to specific sequences of nucleic acids by application of fluorescence resonance energy transfer

We show a new application of fluorescence resonance energy transfer (FRET) in two stages to detect specific sequences of nucleic acids. In the first stage, two fluorescently tagged oligonucleotides hybridize with a complementary target molecule to produce FRET. The sequences of the oligonucleotides and spectral properties of fluorophores are chosen to provide a basis for an efficient energy transfer. In the next step, the specificity of hybridization is tested by competition of labeled probes with an excess of unlabeled oligonucleotides of the same sequence. The resulting emission spectra, one obtained in the excess of unlabeled donor probe and the other produced in the excess of unlabeled acceptor probe, are compared with the spectrum from the first stage to look for differences in the emission pattern of the fluorescent labels. We show that it is possible to detect the existence of specific hybrids composed of the two probes and complementary target molecule even in very unfavorable conditions, such as the presence of unhybridized probes in the final reaction mixture, secondary nonacceptor quenching of donor probe fluorescence, and strong background emission of acceptor produced by its direct excitation with a donor excitation light.     

Pyrene-modified guanosine as fluorescent probe for DNA modulated by charge transfer

8-(Pyren-1-yl)-2'-deoxyguanosine (Py-G) was incorporated synthetically as a modified DNA base and optical probe into oligonucleotides. A variety of Py-G-modified DNA duplexes have been investigated by methods of optical spectroscopy. The DNA duplex hybridization can be observed by both fluorescence and absorption spectroscopy since the Py-G group exhibits altered properties in single strands versus double strands for both spectroscopy methods. The fluorescence enhancement upon DNA hybridization can be improved significantly by the presence of 7-deazaguanin as an additional modification and charge acceptor three bases away from the Py-G modification site. Moreover, Py-G in DNA can be applied as a photoinducable donor for charge transfer processes when indol is present as an artificial DNA base and charge acceptor. Correctly base-paired duplexes can be discriminated from mismatched ones by comparison of their fluorescence quenching.     

The reassociation of factor Va from its isolated subunits

Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl-2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.     

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells.

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.     

The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.     

Scanning near-field fluorescence resonance energy transfer microscopy

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.     

Role of metal ions in the T- to R-allosteric transition in the insulin hexamer.

The role of metal ions in the T- to R-allosteric transition is ascertained from the investigation of the T- to R-allosteric transition of transition metal ions substituted-insulin hexamers, as well as from the kinetics of their dissociation. These studies establish that ligand field stabilization energy (LFSE), coordination geometry preference, and the Lewis acidity of the metal ion in the zinc sites modulate the T- to R-state transition. (1)H NMR, (113)Cd NMR, and UV-vis measurements demonstrate that, under suitable conditions, Fe2+/3+, Ni2+, and Cd2+ bind insulin to form stable hexamers, which are allosteric species. (1)H NMR R-state signatures are elicited by addition of phenol alone in the case of Ni(II)- and Cd(II)-substituted insulin hexamers. The Fe(II)-substituted insulin hexamer is converted to the ferric analogue upon addition of phenol. For the Fe(III)-substituted insulin hexamer, appearance of (1)H NMR R-state signatures requires, additionally to phenol, ligands containing a nitrogen that can donate a lone pair of electrons. This is consistent with stabilization of the R-state by heterotropic interactions between the phenol-binding pocket and ligand binding to Fe(III) in the zinc site. UV-vis measurements indicate that the (1)H NMR detected changes in the conformation of the Fe(III)-insulin hexamer are accompanied by a change in the electronic structure of the iron site. Kinetic measurements of the dissociation of the hexamers provide evidence for the modulation of the stability of the hexamer by ligand field stabilization effects. These kinetic studies also demonstrate that the T- to R-state transition in the insulin hexamer is governed by coordination geometry preference of the metal ion in the zinc site and the compatibility between Lewis acidity of the metal ion in the zinc site and the Lewis basicity of the exogenous ligands. Evidence for the alteration of the calcium site has been obtained from (113)Cd NMR measurements. This finding adds to the number of known conformational changes that occur during the T- to R-transition and is an important consideration in the formulation of allosteric mechanisms of the insulin hexamer.     

Structural transition in the metal-free hexamer of protein-engineered [B13 Gln]insulin

For hexamer formation of native insulin the repulsive potential of six B13 Glu carboxylate groups coming together in the centre is overcome by zinc binding to B10 His. Substitution of Gln for Glu in position B13 by site-directed mutagenesis, i.e. replacement of the repelling carboxylates by amide groups, which are offering H-bonding potential, enhances association and allows a metal-free hexamer to form. Merely upon addition of zinc ions this hexamer undergoes the T6----T3R3 respectively T6----R6 structural transition which in the native 2Zn insulin hexamer is inducible only by additives like inorganic anions or phenolic compounds. [B13 Gln]Insulin hexamers are transformed by phenolic compounds, but not by anions, even in the absence of any metal. The structural transformation of insulin can thus be brought about in two ways: By inorganic ions with the zinc ions as their points of attack, which preexist in the nontransformed hexamer, and by phenol, for which the binding sites close to the B5 histidines come into existence only with the transformation. Therefore transformed and non-transformed hexamers, i.e. molecules with helical and extended B chain N-terminus, must be related in a dynamic equilibrium. Phenol acts as a wedge jamming the structure in the transformed state and trapping the zinc ions. Combination of transformed 2Zn[B13 Gln]insulin and metal-free native insulin in the absence of additives results in a redistribution of the zinc ions in favour of native insulin which is an outcome of the dynamic equilibrium and also demonstrates an influence of B13 charge on metal binding affinity. Transformation of a single subunit in a hexamer would lead to bad contacts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hg2+, Cu2+, and Pb2+ -induced changes in Photosystem II photochemical yield and energy storage in isolated thylakoid membranes: A study using simultaneous fluorescence and photoacoustic measurements

Simultaneous fluorescence and photoacoustic measurements have been used to study the effects of metal ions (copper, lead, and mercury) during dark incubation of thylakoid membranes. The values of the chlorophyll fluorescence parameters Fo (initial fluorescence yield with the reaction centers in the open state), Fm (maximal fluorescence yield), Ft (steady state fluorescence yield) and the calculated parameters, _o (maximal quantum yield of Photosystem II photochemistry) and _t (actual quantum yield of Photosystem II photochemistry), strongly decreased in the presence of the metal ions coinciding with an increase in the non-photochemical deexcitation rate constant k(N). It was observed that photosynthetic energy storage measured by photoacoustic spectroscopy also decreased but a large portion of energy storage remained unaffected even at the highest metal ion concentrations used. A maximal inhibition of photosyntheti c energy storage of 80% and 50% was obtained with Hg²⁺ and Cu²⁺-treated thylakoids, respectively, while energy storage was insensitive to Pb²⁺. The results are consistent with the known predominant inhibition of the donor side of Photosystem II by the metal ions. The insensitive portion of energy storage is attributed to the possible recurrence of cyclic electron transport around Photosystem II that would depend on the extent of inhibition produced on the acceptor side by the metal ion used.     

Types of interaction of meso‐tetraphenylporphyrin with alkali and alkaline‐earth metal ions

The cavity in a porphyrin can accommodate metal ions through electron donor–acceptor (EDA) interaction in acetonitrile media without any specially designed fabrication with the porphyrin subunit. Alkali metal ion forms a complex with meso‐tetraphenylporphyrin (TP) in 2:1 stoichiometry, while the bivalent Mg²⁺ ion follows a 1:1 stoichiometry. A fluorescence interaction study indicated that TP can behave like a chemosensor for these ions present in the blood electrolytes. Specifically, for the alkali metal ions intensity‐based sensing was observed, due to inhibition of photoinduced electron transfer (PET), entailing enhancement of fluorescence intensity, and for the alkaline‐earth Mg²⁺ a mixed quenching was observed. Na⁺ and K⁺ ions can be differentiated depending upon the extent of fluorescence enhancement. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f_D) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f_D percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f_D value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f_D (mf_D) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f_D analysis.     

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.     

Analysis of coupled bimolecular reaction kinetics and diffusion by two-color fluorescence correlation spectroscopy: enhanced resolution of kinetics by resonance energy transfer

In two-color fluorescence correlation spectroscopy (TCFCS), the fluorescence intensities of two fluorescently-labeled species are cross-correlated over time and can be used to identify static and dynamic interactions. Generally, fluorophore labels are chosen that do not undergo F?rster resonance energy transfer (FRET). Here, a general TCFCS theory is presented that accounts for the possibility of FRET between reactants in the reversible bimolecular reaction, [reaction: see text] where k(f) and k(b) are forward and reverse rate constants, respectively (dissociation constant K(d) = k(b)/k(f)). Using this theory, we systematically investigated the influence on the correlation function of FRET, reaction rates, reactant concentrations, diffusion, and component visibility. For reactants of comparable size and an energy-transfer efficiency of approximately 90%, experimentally measurable cross-correlation functions should be sensitive to reaction kinetics for K(d) > 10(-8) M and k(f) >or= approximately 10(7) M(-1)s(-1). Measured auto-correlation functions corresponding to donor and acceptor labels are generally less sensitive to reaction kinetics, although for the acceptor, this sensitivity increases as the visibility of the donor increases relative to the acceptor. In the absence of FRET or a significant hydrodynamic difference between reactant species, there is little effect of reaction kinetics on the shape of auto- and cross-correlation functions. Our results suggest that a subset of biologically relevant association-dissociation kinetics can be measured by TCFCS and that FRET can be advantageous in enhancing these effects.     

Switch between charge transfer and local excited states in 4-aminophenyl-substituted Hantzsch 1,4-dihydropyridine induced by pH change and transition metal ions.

The absorption and fluorescence spectra of a Hantzsch 1,4-dihydropyridine derivative bearing a N,N-dimethylaminophenyl group at 4-position (H(2)Py-PhN(CH(3))(2)) in aprotic solvents have been examined and compared to model compounds 4-phenyl- and 4-methyl-substituted Hantzsch 1,4-dihydropyridines (H(2)Py-Ph and H(2)Py-Me). While H(2)Py-Ph and H(2)Py-Me show fluorescence around 420 nm from the local excited state of the dihydropyridine chromophore, H(2)Py-PhN(CH(3))(2) exhibits fluorescence around 520 nm from the intramolecular charge transfer (ICT) state involving the aniline and dihydropyridine groups as donor and acceptor, respectively. Upon addition of an acid to the solution of H(2)Py-PhN(CH(3))(2), the amino group in the aniline is protonated. Thus, the photoinduced intramolecular charge transfer is prevented, and only the fluorescence from the local excited state of the dihydropyridine chromophore can be detected. These changes in the fluorescence behavior are fully reversible: subsequent addition of a base to the acidic solution leads to the recovery of the ICT fluorescence and the quenching of the local fluorescence. Transition metal ions also can switch the fluorescence of H(2)Py-PhN(CH(3))(2). Evidence for the interaction between transition metal ions and the amino group in the dimethylaniline have been provided by absorption and emission spectrum as well as NMR studies.     

FLIM on a wide field fluorescence microscope

Summary FLIM (Fluorescence Lifetime Imaging Microscopy) is a new tool to detect interaction between proteins. The proteins under investigation are fused with fluorescent donor and acceptor molecules. Interaction between the two proteins is accompanied by direct energy transfer from donor to acceptor (FRET), resulting in a shorter lifetime of the fluorescence emitted by the donor molecule. This change in lifetime is detected by FLIM. Fluorescence lifetime imaging can now be done on a widefield fluorescence microscope by using an attachment that is easy to install and simple to operate. The new LIFA attachment is equipped to use different excitation sources. High brightness modulated LEDs as well as lasers modulated by an Accousto Optical Modulator can be used as excitation light source. A modulated image intensifier with digital camera is used as a detector. Power supplies and signal generator are integrated in one control unit that is connected to the light source, detector and computer. All parameters for image acquisition, processing and viewing are easy accessible in the user interface of the software package that uses a modular structure. Lifetime images showing FRET in MCF7 cells with ErbB1-GFP as donor and Py72/Cy3 as acceptor that were taken at EMBL, Heidelberg are shown.     

Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity

Factor VIII circulates as a divalent metal ion-dependent heterodimer comprised of a light chain (LC) and a heavy chain (HC). Reassociation of factor VIII subunits was assessed using fluorescence energy transfer where LC and HC were labeled with acrylodan (Ac; fluorescence donor) and fluorescein-5-maleimide (Fl; fluorescence acceptor), respectively. The reduction of donor fluorescence due to the acceptor was used as an indicator of binding. Subunits associated with high affinity (K(d) = 53.8 nM) in the absence of metal ion and presence of EDTA. However, this product showed no cofactor activity, as measured by a factor Xa generation assay. In the presence of 25 mM Ca(2+), no increase in the intersubunit affinity was observed (K(d) = 48.7 nM) but specific activity of the cofactor was approximately 30% that of native factor VIII. At saturating levels of Fl-HC relative to Ac-LC, donor fluorescence decreased to 79.3 and 73.5% of its original value in the absence and presence of Ca(2+), respectively. Thrombin cleaved the heterodimers that were associated in the absence or presence of Ca(2+) with similar efficiency, indicating that the lack of activity was not the result of a defect in activation. Cu(2+) (0.5 microM) increased the intersubunit affinity by approximately 100 fold (K(d) = 0.52 nM) and the specific activity to approximately 60% of native factor VIII. The former effect was independent of Ca(2+), whereas the latter effect required Ca(2+). These results indicate that the intersubunit association in factor VIII is primarily metal-ion independent while divalent metal ions serve specific roles. Ca(2+) appears essential to promote the active conformation of factor VIII while Cu(2+) primarily enhances the intersubunit affinity.