Volume 382, Issue 3

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore coated beads that mimic the behaviour of multiliganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Fluorescence correlation experiments were performed on rhodamine 6G in PDMS spincoated films on glass surfaces. With polarised excitation, ensemble bleaching of the dye and single molecule intensity fluctuations were observed. From the statistics of single molecule intensity data taken at different positions in the film, correlation functions were calculated. Two modes of motion with exponential decay shapes and correlation times of τ = 0.15 s and τ = 0.7 s could be detected. Potential origins of intensity fluctuations are lateral diffusion, rotational diffusion or intramolecular fluctuations of dyes involving spectral diffusion or photoinduced processes. From the experimental results, lateral diffusion can be ruled out as a motional mode. Single molecule fluctuations are assigned to changes of the molecular configuration of the dyes, which are rigidly bound to the glass. To assess the environmental influence on such molecular motions, the bulk viscosity of the PDMS was varied over two orders of magnitude, leading to changes of τ of the slow mode by a factor of four. This result proves the sensitivity of the single molecule fluctuations to the molecular scale dynamics of the surrounding polymer matrix and makes the correlation time a measure of the local environment of dye probes.

We investigated the specific binding of epidermal growth factor (EGF) to its membranebound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodaminelabeled EGF (RhEGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace RhEGF binding, demonstrating no crossreaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K of 1.5 x 10[9]M[-1]. The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k = 2.9 x 10[-4] s[-1]. The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligandreceptor interactions in cell cultures. This is a step forward toward largescale drug screening in cell cultures.

The interactions between a cationic polymer, poly(2- dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodaminelabeled 25-mer phosphodiester oligonucleotides (RhONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh ON complexes was investigated as a function of the charge ratio (+/) by increasing the pDMAEMA concentration and keeping the RhON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/RhON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al., 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several RhONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. 1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i. e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (e), as revealed from PCH analysis, was greater than e for the free Rh ONs, indicating that the RhONs were attached to pDMAEMA chains.

The microtubule cytoskeleton plays an important role in eukaryotic cells, e. g., in cell movement or morphogenesis. Microtubules, formed by assembly of tubulin dimers, are dynamic polymers changing randomly between periods of growing and shortening, a property known as dynamic instability. Another process characterizing the dynamic behaviour is the socalled treadmilling due to different binding constants of tubulin at both microtubule ends. In this study, we used tetramethylrhodamine (TMR)labeled tubulin added to microtubule suspensions to determine the net exchange rate (NER) of tubulin dimers by fluorescence correlation spectroscopy (FCS) as a measure for microtubule dynamics. This approach, which seems to be suitable as screening system to detect compounds influencing the NER of tubulin dimers into microtubules at steadystate, showed that taxol, nocodazole, colchicine, and vinblastine affect microtubule dynamics at concentrations as low as 10[-9] 10[-10] M.

We have built a fluorescence correlation spectroscopy (FCS) microscope for ultraviolet excitation (280 300 nm) and emission. With UV excitation the fluorescence of natural fluorophores such as the modified nucleotide 2-aminopurine can be analyzed. The sensitivity of a natural fluorophore toward conformational changes can reveal dynamics in biomolecules. UVFCS is well suited for detection of intensity fluctuations related to such conformational dynamics. Here we show UVFCS measured on pQuarterphenyl and on 2-aminopurine (2-AP). The triplet state rate constants and the excitation cross section for 2- AP were estimated to k[23]=1 x 10[6] s[-1], k[31]=3 x 10[5] s[-1], and σ=2 x 10[-17] cm[2].

CysteinyltRNA synthetase catalyzes the addition cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyltRNA synthetase. In this study, we sought to clone and express the fulllength gene for the human cytoplasmic cysteinyltRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxylterminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a fulllength cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyltRNA synthetases in the carboxylterminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyltRNA synthetase contained insertion of 8 bases in the 3 coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results the replacement of the carboxyterminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the fulllength and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA with cysteine.

The calcium ionophore A23187, which was used to i n c rease the intracellular calcium concentration ([Ca[2+]]), was analyzed for effects on bovine leukemia virus (BLV) expression in two BLV infected cell lines. To clarify the role of intracellular free calcium in this response, [Ca[2+]] was measured during ionophore treatment with the fluorescent calcium indicator Fura-2. Elevation of intracellular calcium under these conditions caused an enhancement of BLV gp51 and p24 synthesis as well as an activation of the BLV long terminal repeat (LTR) in a dosedependent manner. Furthermore, it was observed that elevated levels of intracellular calcium following A23187 stimulation lead to activation of NFκB . Based on inhibitor studies, we hypothesize that the effect of A23187 on BLV expression appears to be mediated by PKC.

2-N[3]SLATP [2-azido-2,3O(1-oxyl-2,2,5,5-tetramethyl 3-carbonylpyrroline) adenosine triphosphate], a photoaffinity spinlabeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotidebinding site stoichiometry of sarcoplasmic reticulum (SR) Ca[2+]ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spinspin interactions between two radicals corresponding to a distance of about 15 å between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca[2+]ATPase is a dimer with the nucleotidebinding sites in close proximity.

The mechanism of interaction of the lipopolysaccharide (LPS)binding protein, LBP, with differently composed symmetric and asymmetric planar lipid bilayers was investigated in electrical measurements (membrane current, potential, capacitance). From a change of the inner membrane potential difference, binding of LBP to membranes was deduced. After addition of LBP to one side of the membrane, binding of antiLBP antibodies and LPS to LBP on both sides of the bilayer was observed. Effects resulting from an interaction of antiLBP antiserum with membranebound LBP depend on the side of addition of the antiserum, indicating a directed intercalation of LBP into the membrane. Addition of LPS to the same side as LBP may induce a change of the conformation of LBP or its orientation in the membrane. Based on these observations, we propose that LBP intercalates in a directed orientation into negativelycharged membranes and assumes a transmembrane configuration. Moreover, preincubated complexes of LPS and LBP do not interact with membranes. These experiments show that reconstituted planar membranes are a suitable tool for investigations of the interaction of non poreforming proteins that are involved in signal transduction.

The urokinase plasminogen activator receptor (uPAR) is a membrane protein active in localizing the plasminogen activation cascade system on the cell surface. The resulting pericellular proteolytic activity is responsible for degradation reactions in the extracellular matrix that are needed for the invasion of cancer cells, thus making uPAR a potential target for antiinvasive therapy based on binding antagonists. A remarkable property of the uPAuPAR system is a pronounced species specificity in ligand recognition. We have now cloned and studied uPAR from four primate species and show that even though these sequences contain very few substitutions relative to the human uPAR, the receptor protein products differ markedly in terms of ligand selectivity. Thus, a well described competitive peptide antagonist directed against the human uPAR reacts with only one of the monkey receptors (chimpanzee uPAR), in spite of the fact that uPAR from all of the four species crossreacts with human uPA. Notably, uPAR from African green monkey, which is completely devoid of reactivity with the peptide, contains only three substitutions relative to chimpanzee uPAR in the molecular regions critical for binding. These findings aid the elucidation of the structure/function relationship of uPAR and, unexpectedly, identify a structural distinction governing the binding of uPA and a very similar peptide antagonist.

Using sitedirected mutagenesis we deleted the two interchain disulfide bonds of insulin, separately or both, by substitution of the cysteine residues with serine. Deletion of A20-B19 or both of the two interchain disulfide bonds resulted in the complete loss of secretion of the mutant singlechain porcine insulin precursor (PIP) from Saccharomyces cerevisiae cells. Removal of the A7-B7 disulfide bond resulted in a large reduction of secretion, but we could obtain the mutant for analysis of its biological and some physico chemical properties. The A7-B7 disulfide bond deleted insulin mutant retained only 0.1% receptorbinding activity compared with porcine insulin, and its in vivo biological potency measured by mouse convulsion assay was also very low. We also studied some physicochemical properties of the mutant using circular dichroism, native polyacrylamide gel electrophoresis and reversedphase HPLC, which revealed some structural changes of the mutant peptides compared to native insulin. The present study shows that the two interchain disulfide bonds are important for efficient in vivo folding/secretion of PIP from yeast, especially the A20-B19 disulfide bond, and that the A7-B7 disulfide bond is crucial for maintaining the native conformation and biological activity of insulin.

Tricorn protease is believed to act downstream of the proteasome, or of other ATPdependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricornlike genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRIScC77 and TRIScE87) are very similar in their PDZ and TSP domains, but rather divergent in their βpropeller domains. We have expressed one of them, TRIScC77, in E. coli and characterized the recombinant protein structurally and functionally. TRIScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coli and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. Thefact that Tricorn like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin type peroxidases by the bisglutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3Dstructures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the histagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solventexposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the [1]Nglutathionyl residue of trypanothione, as evidenced by the structure of [1]Nglutathionylspermidinederivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cisPro111 (110)bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXNsubstrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the [1]Nglutathionylspermidine binding in the CfTXN2H6-Cys44Ser structure.

We present results of an approach in which lowdensity labeled DNA itself provides an amplification of the crosscorrelated fluorescent signal in the twocolor crosscorrelation function. Tetramethylrhodamine-4- dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp t a rget DNA. We call this novel approach the twocolor FCS signal amplification. The signal amplification is an example for interactions of two ligands with diff e rent colors at multiple positions of the same target.

Fluorescence correlation spectroscopy is an attractive tool for monitoring molecular interactions in solution. We report here a new and highly sensitive method for studying the interaction of aptamers with their targets using this technique. In vitro selection technology is a combinatorial method for the generation of nucleic acid receptors (aptamers) that are capable of binding to various target molecules. Using the in vitro selection approach we isolated RNAs which bind to the antibiotic moenomycin with high affinity. The formation of RNAmoenomycin complexes was studied by fluorescence correlation spectroscopy with a tetramethylrhodaminelabeled derivative of moenomycin.

Late endosomes accumulate internal membranes within the lumen of the organelle. These internal membranes are enriched in the late endosome specific phospholipid, lysobisphosphatidic acid (LBPA). The organization of LBPArich membrane domains is not well characterized. Using an LBPAspecific monoclonal antibody (6C4), we show that these membrane domains are not accessible from the cytoplasm. Using fluorescence correlation spectroscopy, we also show that 6C4 only binds sonicated, but not intact, late endosomes, presumably reflecting the release of internal membranes upon endosome rupture.

Fluorescence Correlation Spectroscopy (FCS) offers the possibility to measure molecular interactions between active compounds and drug delivery systems such as cationic peptides or polymeric nanoparticles. In order to investigate the potential of FCS for drug carrier design, a complex made of protamine, a cationic peptide, and a 19mer oligonucleotide was characterised. Protamine was used to form proticles, agglomerates consisting of the oligonucleotide and the cationic peptide. The binding kinetics and proticle formation was studied by FCS. Complete binding of the oligonucleotide to protamine was achieved at a 1:2.5 (w/w) ratio. From the diffusion coefficient, D, a mean value for the hydrodynamic diameter was calculated at 53 nm, which was in agreement with data obtained from photon correlation spectroscopy (PCS). Oligonucleotide loading into cationic monomethylaminoethylmethacrylate (MMAEMA) nanoparticles was also determined by this method at 5.6 % (5.6 g per 100 g of nanoparticles).

Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuations in the fluorescence emission of small molecular ensembles, thus providing information about a multitude of parameters, such as concentrations, molecular mobility and dynamics of fluorescently labeled molecules. Performed within diffractionlimited confocal volume elements, FCS provides an attractive alternative to photobleaching recovery methods for determining intracellular mobility parameters of very low quantities of fluorophores. Due to its high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescence flickering. Furthermore, if molecular mobility is to be probed, nonspecific interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as certain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likely to nonspecifically interact with cellular substructures.

Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.

Radicicol (RAD) and the benzoquinone ansamycin geldanamycin (GA) are potential anticancer drugs known to inhibit heat shock protein 90 (hsp90) and, therefore, the activation of proteins dependent on its function such as protooncogenic kinases and nuclear receptors. Using the glucocorticoid receptor (GR) as a model system we analysed the effects of RAD and various benzoquinone ansamycins. All compounds efficiently abolished GRdependent transactivation. Surprisingly, whenever one of the ansamycins was applied in combination with RAD, synergistic inhibition of GRdependent transcription and of hormone binding of GR was observed. In contrast, combination of two ansamycins showed no synergy. These findings suggest synergism within the hsp90 dimer and may open new ways to explore hsp90 as therapeutic target.