Rapid kinetic studies on calcium interactions with native and fluorescently labeled calmodulin

Stopped-flow studies on calcium binding to calmodulin showed that under pseudo first order conditions the reaction was complete within 2.5 milliseconds. The time course for calcium dissociation from the native protein showed a single kinetic phase (τ1^?1 = 10S^?1) while that from the dansylated derivative revealed a second slower kinetic phase (τ1^?1 = 10S^?1, τ2^?1 = 0.31S^?1) that accounted for about one-half of the total fluorescence decrease. Therefore the dansyl derivative of calmodulin may provide a useful tool for studying conformational changes in the protein not reflected by the active site tyrosines.     

Fusion of fluorescently labeled Sendai virus envelopes with living cultured cells as monitored by fluorescence dequenching

Fluorescently labeled (bearing N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine (N-NBD-PE)) reconstituted Sendai virus envelopes (RSVE) were used to study fusion between the viral envelopes and cultured living cells such as lymphoma, Friend erythroleukemia cells (FELC) and L cells. Incubation of fusogenic viruses with the above cell lines resulted in a relatively high degree (40-45%) of fluorescence dequenching. On the other hand, incubation of unfusogenic (trypsin or phenylmethylsulfonylfluoride (PMSF)-treated) RSVE with these cells led to very little (6-9%) fluorescence dequenching. The degree of fluorescence dequenching was linearly correlated to the surface density of the virus-inserted N-NBD-PE molecules. Fluorescence photobleaching recovery experiments showed that fusion of fluorescent RSVE with FELC resulted in an infinite dilution of the fluorescent molecules in the recipient cell membranes. The fluorescent probe 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (N-NBD-Cl) was covalently attached to envelopes of intact Sendai virions without significantly impairing their biological activity. Incubation of fluorescently labeled, intact Sendai virions with cultured cells resulted in about 20% fluorescence dequenching. The present data clearly indicate that fluorescently labeled Sendai virions can be used for a quantitative estimation of the degree of virus-membrane fusion.     

Evaluation of fluorescently labeled xylopyranosides as probes for proteoglycan biosynthesis

A new fluorescent analog to the antiproliferative 2-(6-hydroxynaphthyl)-beta-d-xylopyranoside has been synthesized and tested on a T24 cell line. The new analog was efficiently uptaken by the T24 cells but did not initiate priming of GAG chains. The results are similar to other fluorescently labeled analogs and we propose that these compounds are too large and unpolar to efficiently function as GAG-primers.     

Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies

Polyethyleneimine (PEI) has been used previously as a nonviral DNA transfer vector. In this article, we demonstrate its use as a vehicle for transmembrane delivery of proteins in cell culture conditions. Linking proteins to PEI required no other treatment beyond mixing them with PEI. The bond between PEI and protein combined at optimal ratios was maintained in electrophoresis, even in the presence of 2.5% sodium dodecyl sulfate (SDS). The optimal time for delivery of proteins was determined to be 24 h. We have successfully delivered an Alexa 488-labeled avidin protein into human glioblastoma cells. A functional antibody against the nuclear protein lamin was delivered into human fibroblasts and reacted with lamin inside live cells. PEI-based delivery of antibodies and fluorescently labeled proteins can be used for fluorescent detection, tracking, and evaluation of cellular protein function in vivo.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative

The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.     

Synthesis and biophysical studies on fluorescently labeled oligodeoxyribonucleotides

Two highly fluorescent compounds, viz. 6-(6-isobutyrylamino-1,3-dioxo-1 H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid and 6-(6-dimethylamino-1,3-dioxo-1 H,3H-benzo[de]isoqu-inolin-2-yl)-hexanoic acid have been synthesized, characterized, and attached to 12-mer oligodeoxyribonucleotides at their 5'-end using suitable linker molecule. These labeled oligodeoxyribonucleotides have shown appreciable fluorescence even at 0.0019 microM concentrations. Thermal denaturation studies have shown comparatively higher Tm values when oligodeoxyribonucleotides are labeled. These labeled oligodeoxyribonucleotides have been purified on RP-HPLC utilizing their hydrophobicity and on polyacrylamide gel because of their easy detection due to fluorescence.     

Microtubule-independent motility and nuclear targeting of adenoviruses with fluorescently labeled genomes

A novel adenovirus system for analyzing the adenovirus entry pathway has been developed that contains green fluorescent protein bound to the encapsidated viral DNA (AdLite viruses). AdLite viruses enter host cells and accumulate around the nuclei and near the microtubule organizing centers (MTOC). In live cells, individual AdLite particles were observed trafficking both toward and away from the nucleus. Depolymerization of microtubules during infection prevented AdLite accumulation around the MTOC; however, it did not abolish perinuclear localization of AdLite particles. Furthermore, depolymerization of microtubules did not affect AdLite motility and did not affect gene expression from wild-type adenovirus and adenovirus-derived vectors. These data revealed that adenovirus intracellular motility and nuclear targeting can be supported by a mechanism that does not rely on the microtubule network.     

Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin

Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], association with MgADP decreased the probe fluorescence by 30%, whereas binding to actin increased the emission by a factor of 2. In the ternary complex acto-S1-AF X MgADP, the effect of nucleotide on the intensity of the attached fluorescein canceled the effect of actin. The fluorescence state of this ternary complex was similar to that of S1-AF X MgADP. The emission of S1-AF was resolved into two components with lifetimes of 4.3 and 0.6 ns and relative contributions of 33% and 67%, respectively. Interaction of S1-AF with nucleotides and actin did not alter the lifetimes but significantly shifted their fractional contributions. Quenching studies showed that the short lifetime likely arose from the fluorescein moiety statically quenched by internal groups. Binding of MgADP to the salicylate derivative [S1 modified with 5-(iodoacetamido)salicylic acid at SH1 (S1-SAL)] induced a 25% enhancement of the probe fluorescence, whereas formation of acto-S1-SAL decreased the emission by 10% regardless of whether MgADP was bound to the protein. Both labeled S1 species bound MgADP with a similar affinity, comparable to that of unmodified S1 previously reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new approach to SNP genotyping with fluorescently labeled mononucleotides

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3′ end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.     

Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases

Activation of caspases is the key event of apoptosis and different approaches were developed to assay it. To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.     

Interaction of fluorescently labeled pardaxin and its analogues with lipid bilayers.

Fluorescence measurements were used to monitor the interaction of the neurotoxin pardaxin and its analogues with membranes. Eight peptides were selectively labeled with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole-4-yl, either at their N-terminal or at their C-terminal. No detectable changes in membrane permeability or hemolytic activity were observed upon modification. Upon the titration of solutions containing the different peptides with small unilamellar vesicles, the fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled pardaxin and its analogues, but not those of control peptides, displayed blue shifts in addition to enhanced intensities upon relocation of the probe to a more apolar environment. The results revealed that the N terminus of pardaxin is buried within the lipid bilayer while the C terminus is located at the bilayer's surface. Binding isotherms were obtained from the observed increases in the fluorescence emission yields, from which surface partition constants, in the range of 10(4) M-1, were in turn derived. The existence of an aggregation process was suggested by the shape of the binding isotherms. Furthermore, the results show good correlation between the incidence of aggregation and the ability of the different analogues to induce the release of relatively large molecules from vesicles. As such, our results suggest that the mechanism of pore formation employed by pardaxin and its analogues could be described by the "barrel stave" model.     

Interactions of a fluorescently labeled peptide with kringle domains in proteins

The tripeptide Lys-Cys-Lys has been synthesized and covalently labeled at the cysteine sulfhydryl with 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid to produce a fluorescent labeled peptide (FLP). When excited at 340 nm, the FLP fluoresces strongly with maximal intensity at 405 nm. Addition of proteins containing the kringle lysine-binding domain, such as human lipoprotein (a) and plasminogen kringle 4, significantly attenuate the fluorescence intensity of the FLP. Other proteins, such as bovine serum albumin, did not affect the quantum yield of FLP fluorescence. When human lipoprotein (a) is bound to a lysine-Sepharose affinity column, FLP was found to effectively elute the protein, indicating that the peptide can compete with lysine for the kringle-binding site on lipoprotein (a). The data suggest that FLP binds specifically to kringles through the lysine residues on the peptide, and that binding significantly affects the fluorescence from the labeled peptide. These properties of FLP make it a potentially useful tool for studying the relative affinity of different kringles for lysine binding, which is thought to be an important mechanism for kringle-target protein interactions.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes.

Dermaseptin, a 34 amino-acid residue antimicrobial polypeptide [Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., & Nicolas, P. (1991) Biochemistry 30, 8824-8830] was synthesized and selectively labeled at its N-terminal amino acid with either 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD), rhodamine, or fluorescein. The fluorescent emission spectra of the NBD-labeled dermaseptin displayed a blue-shift upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. Titrations of solutions containing NBD-labeled dermaseptin with SUV composed of zwitterionic or acidic phospholipids were used to generate binding isotherms, from which were derived surface partition constants of (0.66 +/- 0.06) x 10(4) M-1 and (2.8 +/- 0.3) x 10(4) M-1, respectively. The shape of the binding isotherms, as well as fluorescence energy transfer measurements, suggests that some aggregation of membrane-bound peptide monomers occurs in acidic but not in zwitterionic vesicles. The preferential susceptibility of the peptide to proteolysis when bound to zwitterionic but not to acidic SUV suggests that these aggregates might then penetrate a relatively short distance into the hydrophobic region of the acidic membrane. Furthermore, the results provide good correlation between the peptide's strong binding and its ability to permeate membranes composed of acidic phospholipids, as revealed by a dissipation of diffusion potential and a release of entrapped calcein from SUV.     

Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.