Fluorescence Polarization Analysis Research Articles

Genotyping of Apolipoprotein E: Comparative Evaluation of Different Protocols

Disease-associated gene polymorphisms provide both scientific insight into pathophysiological mechanisms and clinical information regarding risk and progression. Of special interest is the epsilon4 allele of the apolipoprotein E gene, which has emerged as a substantial risk factor for late-onset forms of Alzheimer disease and also influences the risk of cardiovascular disease. Genotyping of apolipoprotein E can be performed by several methods; presented here are a quality and cost-benefit analysis of four different protocols on a cohort of 42 clinical samples is included in the unit. Each method resulted in genotyping with a high sensitivity and specificity. The newer microtiter-plate-based high-throughput techniques, fluorescence polarization and SNaPshot analysis, were as reliable as the traditional techniques of restriction fragment length polymorphism analysis and reverse hybridization. The reverse hybridization method tends to be more cost- and time-effective when the number of analyses is limited, although economy of scale favors fluorescence polarization or SNaPshot analysis in larger studies. The latter approaches also provide the flexibility to investigate other polymorphic disease markers.

RBP38, a novel RNA-binding protein from trypanosomatid mitochondria, modulates RNA stability.

We describe here the isolation and characterization of a novel RNA-binding protein, RBP38, from Leishmania tarentolae mitochondria. This protein does not contain any known RNA-binding motifs and is highly conserved among the trypanosomatids, but no homologues were found in other organisms. Recombinant LtRBP38 binds single and double-stranded (ds) RNA substrates with dissociation constants in the 100 nM range, as determined by fluorescence polarization analysis. Downregulation of expression of the homologous gene, TbRBP38, in procyclic Trypanosoma brucei by using conditional dsRNA interference resulted in 80% reduction of steady-state levels of RNAs transcribed from both maxicircle and minicircle DNA. In organello pulse-chase labeling experiments were used to determine the stability of RNAs in mitochondria that were depleted of TbRBP38. The half-life of metabolically labeled RNA decreased from approximately 160 to approximately 60 min after depletion. In contrast, there was no change in transcriptional activity. These observations suggest a role of RBP38 in stabilizing mitochondrial RNA.

Synthesis of complex phosphopeptides as mimics of p53 functional domains.

A complete set of mono-, di- and triphosphorylated peptides comprising amino acids 10-27, the Mdm2 and p300 binding site(s) of p53, with and without a fluorescein label at the N-terminus, was synthesized by step-by-step solid phase synthesis. Fluorescence polarization analysis revealed that phosphorylation at Thr18 decreased binding to recombinant Mdm2 protein compared with the unphosphorylated and the two other single phosphorylated analogues. Unlabelled multiply phosphorylated peptides corresponding to this amino-terminal transactivation domain proved to be powerful tools in analysing the phosphate specificity of existing anti-p53 monoclonal and polyclonal antibodies using direct ELISA. The tetramerization domain of human p53 protein was modelled with a 53 residue-long unlabelled unphosphorylated and Ser315-phosphorylated peptide pair. CD analysis showed similar alpha-helical structures for both peptides and no major difference in the secondary structure could be observed upon phosphorylation. Size-exclusion HPLC indicated that these synthetic oligomerization domain mimics underwent a pH-dependent tetramerization process, but the presence of a phosphate group at Ser315 did not modify the oligomeric state of the 308-360 p53 fragments. Nevertheless, the fluorescein-labelled Ser315 phosphorylated peptide bound to the downstream signalling ligand DNA topoisomerase I protein with slightly higher affinity than did the unphosphorylated analogue.

Analysis of Early Apoptotic Events in Individual Cells by Fluorescence Intensity and Polarization Measurements

Apoptosis is a dynamic process of variable duration. The ability to continuously detect the death process occurring in single or subgroups of cells is therefore very important in identifying apoptotic cells within a complex population. The Individual Cell Scanner (ICS), a multiparametric, multilaser-based scanning static cytometer, was used in the present report for the continuous monitoring of the apoptosis process. Fluorescence intensity (FI), polarization (FP), kinetic measurements, and cluster analysis of subpopulations were carried out utilizing various fluorescent probes. Hydrogen peroxide-induced apoptosis was monitored online in intact live lymphocytes by continuous sequential measurements of intracellular hyperpolarization. Plasma membrane asymmetry, mitochondrial membrane potential, and lysosomal rupture were monitored in individual cells. Cytoplasmic condensations, due to cell shrinkage and early lysosomal rupture, were found to be very early events of apoptosis. The new analytical capabilities suggested here may provide simple and convenient methods for detecting apoptosis from its earlier stages.

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding.

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide binding to DnaK can be correlated with antimicrobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase activity of live bacteria. Remarkably, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of synthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM to the hinge region around the C-terminal helices D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assisted protein folding. The biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent sequence variations of procaryotic and eucaryotic DnaK molecules in the multihelical lid region, our findings pave the road for the design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding include the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.

Rapid and simple detection of PCR product DNA: a comparison between Southern hybridization and fluorescence polarization analysis

The essential aim of this study was to compare two different methods, Southern hybridization and fluorescence polarization (FP) assay. They both detect specific hybridization and were examined using common asymmetric PCR products and probes. FP assay clearly showed the hybridization of probe DNAs with the asymmetric PCR products of their target genes. Southern blot patterns presented excellent consistency with the results of FP assay. In both methods, two types of Shiga toxin (vero toxin) genes held in enterohaemorrhagic Escherichia coli (EHEC) were used as target genes. For detection of the two genes, stx1 and stx2, two respective DNA probes were synthesized. Both in FP assay and in Southern hybridization, the probe for stx1 hybridized only with the product of stx1 and vice versa. The results of the DNA detection using different methods were completely in agreement. Moreover, FP assay makes it possible to detect the hybridization rapidly. In our high NaCl concentration condition, hybridization between the probes and the asymmetric PCR products could be monitored within about 15min.

Measurement of the time course of DNA/RNA hybridization and RNA detection using fluorescence polarization analysis.

Using fluorescence polarization analysis, the time courses of hybridization between probe oligo-DNAs and target RNAs were measured. The RNAs were amplified using the DNA templates of Shiga toxin genes by NASBA (Nucleic Acid Sequence Based Amplification). Two DNA probes were designed for detecting the genes and they rapidly and specifically hybridized with their target RNA sequences. NASBA could be sufficiently used for the combination and DNA/RNA hybridization could be detected in the fluorescence polarization.

Reactivity of peripheral blood lymphocytes to oxidized low-density lipoprotein: a novel system to estimate atherosclerosis employing the Cellscan.

The assumption that atherosclerosis involves an autoimmune response to oxidized LDL (oxLDL) is based on the presence of immunocompetent cells and immunoglobulin deposition in the atherosclerotic lesions by successful immunomodulation of the atherosclerotic process and by inhibition of experimental atherosclerosis by antioxidants. The Cellscan system is a multiparameter laser-based static cytometer that enables repeated monitoring of the fluorescence intensity (FI) and polarization (FP) of individual living cells. Analysis of intracellular fluorescein fluorescence polarization (IFFP) has previously been used to define activated lymphocyte population. In this study, the Cellscan apparatus has been used to monitor cellular response to oxLDL in patients with atherosclerosis and in controls. The FI and FP of fluorescein diacetate (FDA)-labeled peripheral lymphocytes were measured following exposure to oxLDL in vitro. Using cluster analysis we were able to identify subpopulations of cells that were characterized by their FI and FP. Forty-two subjects were studied: 22 patients with severe coronary heart disease and 22 control individuals, either healthy or with other diseases. Fluorescence intensity of fluorescein-labeled peripheral blood lymphocytes (PBL) was markedly decreased upon exposure to high doses (> 25 micrograms/ml) of oxLDL concurrently with an increase in FP. A specific and dose-dependent reduction in FP of the high-intensity cell subpopulations, accompanied by higher FI, was evident in patients with ischemic heart disease upon exposure to low doses of oxLDL (up to 25 micrograms/ml). Maximal depolarization was shown upon triggering with 2 micrograms/ml oxLDL. The polarization ratio (the mean polarization value of the specific cell population with and without activation) obtained for patients' lymphocytes was significantly lower (p < 0.01) than that of the control group (0.936 +/- 0.05 and 1.028 +/- 0.055, respectively). These data suggest that PBL from patients with active ischemic heart disease show an increased reactivity to oxLDL. A 73% positivity rate was found for ischemic heart disease patients compared with 5% in the control subjects. One of the future prospects of this study might be the advent of a simple and rapid noninvasive test that could assess the extent of atherosclerosis, and possibly even the response to therapy, by monitoring the reactivity of PBL to oxLDL.

A unique loop contributing to the structure of the ATP-binding cleft of skeletal muscle myosin communicates with the actin-binding site.

Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677-689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently crosslinked to loop M (Mant-S1), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677-689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational mobility was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-S1 to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677-689) probably results from a conformational change in loop M (677-689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites.

Phosphorylation of the C-terminal sites of human p53 reduces non-sequence-specific DNA binding as modeled with synthetic peptides.

Phosphorylation of the tumor suppressor p53 is generally thought to modify the properties of the protein in four of its five independent domains. We used synthetic peptides to directly study the effects of phosphorylation on the non-sequence-specific DNA binding and conformation of the C-terminal, basic domain. The peptides corresponded to amino acids 361-393 and were either nonphosphorylated or phosphorylated at the protein kinase C (PKC) site, Ser378, or the casein kinase II (CKII) site, Ser392, or bis-phosphorylated on both the PKC and the CKII sites. A fluorescence polarization analysis revealed that either the recombinant p53 protein or the synthetic peptides bound to two unrelated target DNA fragments. Phosphorylation of the peptide at the PKC or the CKII sites clearly decreased DNA binding, and addition of a second phosphate group almost completely abolished binding. Circular dichroism spectroscopy showed that the peptides assumed identical unordered structures in aqueous solutions. The unmodified peptide, unlike the Ser378 phosphorylated peptide, changed conformation in the presence of DNA. The inherent ability of the peptides to form an alpha-helix could be detected when circular dichroism and nuclear magnetic resonance spectra were taken in trifluoroethanol-water mixtures. A single or double phosphorylation destabilized the helix around the phosphorylated Ser378 residue but stabilized the helix downstream in the sequence.

Purification and Characterization of the Active-Site-Mutated Recombinant Human μ-Calpain Expressed in Baculovirus-Infected Insect Cells

Recombinant human μ-calpain whose active site Cys-115 was substituted with Ser was expressed in insect cells using baculovirus system. The mutant μ-calpain, purified using an affinity-column of calpastatin oligopeptides, had no proteolytic activities of autolysis and caseinolysis. The large subunit of the mutant μ-calpain was processed from the 80 kDa form to the 76 kDa form by the wild type calpain, supporting the intermolecular cleavage mechanism of procalpain during activation. Fluorescence polarization analysis revealed that the mutant μ-calpain retained high affinity toward fluorescein-labeled calpastatin domain 1. Fragmentation of the full-length calpastatin by the wild type calpain was enhanced by pre-incubating the inhibitor with the mutant calpain. The recombinant mutant calpain was suggested to retain the integrity of the high ordered structure of the wild type calpain.

Fluorescence polarization study of a salt bridge between a single chain Fv and its antigen ribonuclease a

The interaction between a single chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of the hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp 31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 × 10 7 and 4.7 × 10 6 M −1 in PBS at pH 7.2 and 37°C, respectively. Whereas the affinity constant of D31A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp 31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.

Ionization, partitioning, and dynamics of tryptophan octyl ester: implications for membrane-bound tryptophan residues

The presence of tryptophan residues as intrinsic fluorophores in most proteins makes them an obvious choice for fluorescence spectroscopic analyses of such proteins. Membrane proteins have been reported to have a significantly higher tryptophan content than soluble proteins. The role of tryptophan residues in the structure and function of membrane proteins has attracted a lot of attention. Tryptophan residues in membrane proteins and peptides are believed to be distributed asymmetrically toward the interfacial region. Tryptophan octyl ester (TOE) is an important model for membrane-bound tryptophan residues. We have characterized this molecule as a fluorescent membrane probe in terms of its ionization, partitioning, and motional characteristics in unilamellar vesicles of dioleoylphosphatidylcholine. The ionization property of this molecule in model membranes has been studied by utilizing its pH-dependent fluorescence characteristics. Analysis of pH-dependent fluorescence intensity and emission maximum shows that deprotonation of the alpha-amino group of TOE occurs with an apparent pKa of approximately 7.5 in the membrane. The fluorescence lifetime of membrane-bound TOE also shows pH dependence. The fluorescence lifetimes of TOE have been interpreted by using the rotamer model for the fluorescence decay of tryptophan. Membrane/water partition coefficients of TOE were measured in both its protonated and deprotonated forms. No appreciable difference was found in its partitioning behavior with ionization. Analysis of fluorescence polarization of TOE as a function of pH showed that there is a decrease in polarization with increasing pH, implying more rotational freedom on deprotonation. This is further supported by pH-dependent red edge excitation shift and the apparent rotational correlation time of membrane-bound TOE. TOE should prove useful in monitoring the organization and dynamics of tryptophan residues incorporated into membranes.

Fluorescence polarization study of a salt bridge between a single-chain Fv and its antigen ribonuclease A

The interaction between a single-chain Fv (sFv) of the monoclonal antibody 3A21 and its antigen, bovine pancreatic ribonuclease A (RNase A), was studied by site-directed mutagenesis of thee hypervariable regions and fluorescence polarization analysis. The affinity constants of wild-type sFv and a mutant sFv D31A (Asp 31 of heavy chain was replaced by Ala) for RNase A were found to be 2.7 × 10 7 and 4.7 × 10 6M −1 in PBS at pH 7.2 and 37°C, respectively. While the affinity constant of D31 A is not affected by NaCl concentration, that of wild-type sFv is almost the same as that of D31A in the presence of more than 1 M NaCl. These results demonstrate that Asp 31 of the heavy chain interacts electrostatically with a positively charged amino acid residue of RNase A.

Calmodulin-Related Changes in Microsomal Membrane Fluidity during Liver Regeneration

Based on our previous findings on the modifying effect of calmodulin (CaM) on the physicochemical properties of biomembrane, we have investigated the possible relationship between intracellular CaM content and endoplasmic reticulum (ER) membrane fluidity and function during liver regeneration. The degree of ER membrane fluidity was estimated by fluorescence polarization analysis with the 1,6-diphenyl-1,3,5-hexatriene probe. Microsomal guanylate cyclase (GC) was used as a functional parameter. The kinetics of the increase in the ER membrane fluidity during liver regeneration was strictly parallel to the CaM surge and was matched by an increase in GC activity. The stimulative effect of splenectomy on liver regeneration and its inhibition by Walker-256 tumor, inferred from the corresponding alterations of CaM levels, were mirrored by the modulation in GC activity. The fluidizing effect of CaM on ER membrane was concluded from the drop in thermotropic transition temperature from 28.3 ± 1.6°C in control membranes to 17.8 ± 1.1°C in membranes from regenerating livers and to 19.8 ± 1.2°C in control membranes treated with CaM. Arrhenius plots of GC activity exhibited a transition temperature of 25.5 ± 1.25°C in controls, which shifted to 20.5 ± 0.9°C in ER membranes from regenerating livers and to 21.7 ± 1.1°C in control membranes treated with CaM. The Hill coefficient for the allosteric activation of the GC by Mn·GTP decreased from 1.49 ± 0.16 in controls to 0.93 ± 0.085 in membranes from regenerating cells and to 0.86 ± 0.073 in CaM-treated membranes. Both effects of CaM were consistent with a fluidity increase in the enzyme's lipid microenvironment. The results of the present study suggest that an early key event in liver regeneration may be the CaM-induced modulation of ER membrane fluidity and function.

Fluorescence polarization analysis of protein-DNA and protein-protein interactions

Fluorescence polarization analysis of protein-DNA and protein-protein interactions

Analysis of Microfibril Orientation in Cotton Fibers by Fluorescence Polarization

Microfibril orientation in a cotton fiber surface is measured by the fluorescence polarization method using microphotometry. To serve as a cotton model, drawn nylon fibers are dyed with a high concentration of fluorescent probe and subjected to a twist tester to obtain certain twisting angles, which are then quantitatively determined from the angular distribution of the polarized fluorescence intensity. For naturally dried cotton fibers, this fluorescence polarization analysis shows the twisting angle to be 24.5°. The method also permits analysis of the twisting sense of fibrils in the surface of cotton fibers without needing to untwist them.

Fluorescence polarization analysis of protein-DNA and protein-protein interactions.

Fluorescence polarization is a powerful technique for characterizing macromolecular associations and can provide equilibrium determinations of protein-DNA and protein-protein interactions. This technique is particularly useful (and better suited than electrophoretic methods) to study low affinity protein-protein interactions. In this review, we have outlined the principles underlying the use of fluorescence polarization to study the assembly of higher order complexes that bind to the CRE. The availability of simple, relatively inexpensive instrumentation means that this technology is no longer only within the realm of the physical biochemist.

Muramyl peptide-binding sites are located inside target cells

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide-binding sites were shown to be located inside T-lymphocytes, macrophages and neuroblastoma cells, but not inside B-cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1-3 cells with K fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D-configuration of isoglutamine residue are important.

In vitro interaction of selected phpspholipid species with mercuric chloride using fourier transform1H-NMR

Many studies on the mercury toxicities have been accumulated since the outbreak of Minamata Disease.' There have been few reports on the reaction mechanisms of mercurials with phospholipids which substantially locate in biological membranes, although the interactions of nucleotides or nucleosides with mercurials have been reported. Recently, the study on the interaction of mercuric chloride (HgCl{sub 2}) with amino polar heads of model membranes containing phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been reported, as the results from the fluorescence polarization analysis using 1,6-diphenyl-1,3,5-hexatriene. The authors demonstrate here the interactions of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) with HgCl{sub 2}, using Fourier transform {sup 1}H-NMR ({sup 1}H-FT-NMR).