Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We designed molecular beacons to detect a point mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, a mutation that has been related to an increased risk for cardiovascular disease and neural tube defects. The application of molecular beacons enables fast, semiautomated, ... More
Flow injection analysis in a microfluidic format.
AuthorsLeach AM, Wheeler AR, Zare RN
JournalAnal Chem
PubMed ID12622393
A microfluidic flow injection analysis system has been designed and evaluated. The system incorporates within a single two-layer poly(dimethylsiloxane) monolith multiple pneumatically driven peristaltic pumps, an injection loop, a mixing column, and a transparent window for fluorescence detection. Central to this device is an injection system that mimics the operation ... More
The pleckstrin homology domain of phospholipase Cbeta transmits enzymatic activation through modulation of the membrane-domain orientation.
AuthorsDrin G, Douguet D, Scarlata S
JournalBiochemistry
PubMed ID16669615
'Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also ... More
Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9799521
'The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a ... More
Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay.
AuthorsBeekman B, van El B, Drijfhout JW, Ronday HK, TeKoppele JM
JournalFEBS Lett
PubMed ID9428733
'Stromelysin-1 (MMP-3) is an important member of the matrix metalloproteinase family. In joint-degrading diseases like arthritis, elevated levels of MMP-3 protein are detected in synovial fluid using immunological methods. However, these methods do not discriminate between active and inactive enzyme. In the present study, a specific stromelysin activity assay was ... More
A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis.
AuthorsMaggiora LL, Smith CW, Zhang ZY
JournalJ Med Chem
PubMed ID1433187
'A general scheme for obtaining a fluorescent donor/acceptor peptide substrate via solid-phase synthesis methodology is presented. The key feature of this method is the design of a glutamic acid derivative that has been modified on the carboxyl side chain with a 5-[(2''-aminoethyl)-amino]naphthelenesulfonic acid (EDANS) to create a fluorescent donor moiety ... More
Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides.
AuthorsTong AK, Ju J
JournalNucleic Acids Res
PubMed ID11861924
'Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide ... More
Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates.
AuthorsTon-That H, Mazmanian SK, Faull KF, Schneewind O
JournalJ Biol Chem
PubMed ID10734144
'Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing ... More
Characterization of the structure and function of W --> F WW domain variants: identification of a natively unfolded protein that folds upon ligand binding.
AuthorsKoepf EK, Petrassi HM, Ratnaswamy G, Huff ME, Sudol M, Kelly JW
JournalBiochemistry
PubMed ID10572009
'The WW domain adopts a compact, three-stranded, antiparallel beta-sheet structure that mediates protein-protein interactions by binding to xPPxY-based protein ligands, such as the PY-ligand (EYPPYPPPPYPSG) derived from p53 binding protein-2. The conserved Trp residues, after which this domain was named, were replaced with Phe so their importance in structural integrity ... More
Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.
AuthorsBlackman MJ, Corrie JE, Croney JC, Kelly G, Eccleston JF, Jameson DM
JournalBiochemistry
PubMed ID12356327
'Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine ... More
Molecular aptamer beacons for real-time protein recognition.
AuthorsLi JJ, Fang X, Tan W
JournalBiochem Biophys Res Commun
PubMed ID11890667
'One of the most pressing problems facing those attempting to understand the regulation of gene expression and translation is the necessity to monitor protein production in a variety of metabolic states. Thus far, there is no easy solution that will either identify or quantitate proteins in real time. Here we ... More
Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9931014
'Phosphatidylinositide-specific phospholipase C-betas play a key role in Ca2+ signaling and are specifically activated by the alphaq family of heterotrimeric G proteins and as well as betagamma subunits. We have determined the affinity between Gbetagamma subunits and GTPgammaS and GDP-liganded Galphaq subunits on membrane surfaces, and their respective affinities to ... More
A continuous assay for DNA cleavage: the application of "break lights" to enediynes, iron-dependent agents, and nucleases.
AuthorsBiggins JB, Prudent JR, Marshall DJ, Ruppen M, Thorson JS
JournalProc Natl Acad Sci U S A
PubMed ID11095715
'Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of "molecular break ... More
A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease.
AuthorsCummings RT, Salowe SP, Cunningham BR, Wiltsie J, Park YW, Sonatore LM, Wisniewski D, Douglas CM, Hermes JD, Scolnick EM
JournalProc Natl Acad Sci U S A
PubMed ID11997440
'A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a ... More
Real-time PCR in the microbiology laboratory.
AuthorsMackay IM
JournalClin Microbiol Infect
PubMed ID15008940
'Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has ... More
Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.
AuthorsEggleson KK, Duffin KL, Goldberg DE
JournalJ Biol Chem
PubMed ID10542284
'The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in ... More
Optimizing primer--probe design for fluorescent PCR.
AuthorsProudnikov D, Yuferov V, Zhou Y, LaForge KS, Ho A, Kreek MJ
JournalJ Neurosci Methods
PubMed ID12581847
'TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then ... More
Design of a Molecular Beacon DNA Probe with Two Fluorophores This work was partially supported by the Office of Naval Research Young Investigator Award N00014-98-1-0621 and by an NSF Career Award (CHE-9733650). We would like to thank Dr. Richard Hogrefe, Paul Imperial, Kelly Christianson, and the Trilink Oligonucleotide Synthesis group for producing the molecular beacons used in this study.
AuthorsZhang P, Beck T, Tan W
JournalAngew Chem Int Ed Engl
PubMed ID11180338
Kinetics of conformational fluctuations in DNA hairpin-loops.
AuthorsBonnet G, Krichevsky O, Libchaber A
JournalProc Natl Acad Sci U S A
PubMed ID9671724
'The kinetics of DNA hairpin-loop fluctuations has been investigated by using a combination of fluorescence energy transfer and fluorescence correlation spectroscopy. We measure the chemical rates and the activation energies associated with the opening and the closing of the hairpin for different sizes and sequences of the loop and for ... More
Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate.
AuthorsPattanaik P, Jain B, Ravindra G, Gopi HN, Pal PP, Balaram H, Balaram P
JournalBiochem Biophys Res Commun
PubMed ID13679069
'Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and ... More
A three-state mechanism for DNA hairpin folding characterized by multiparameter fluorescence fluctuation spectroscopy.
AuthorsJung J, Van Orden A
JournalJ Am Chem Soc
PubMed ID16433541
'The folding of a dye-quencher labeled DNA hairpin molecule was investigated using fluorescence autocorrelation and cross-correlation spectroscopy (FCS) and photon counting histogram analysis (PCH). The autocorrelation and cross-correlation measurements revealed the flow and diffusion times of the DNA molecules through two spatially offset detection volumes, the relaxation time of the ... More
Characterization of the interaction between alphaCP(2) and the 3'-untranslated region of collagen alpha1(I) mRNA.
AuthorsLindquist JN, Kauschke SG, Stefanovic B, Burchardt ER, Brenner DA
JournalNucleic Acids Res
PubMed ID11058131
'Activated hepatic stellate cells produce increased type I collagen in hepatic fibrosis. The increase in type I collagen protein results from an increase in mRNA levels that is mainly mediated by increased mRNA stability. Protein-RNA interactions in the 3''-UTR of the collagen alpha1(I) mRNA correlate with stabilization of the mRNA ... More
Real time detection of DNA.RNA hybridization in living cells.
AuthorsSokol DL, Zhang X, Lu P, Gewirtz AM
JournalProc Natl Acad Sci U S A
PubMed ID9751701
'Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized "molecular beacon" (MB) reporter oligodeoxynucleotides with matched fluorescent donor and acceptor chromophores on their 5'' and 3'' ends. In the absence ... More
Homogeneous time-resolved fluorescence quenching assay (LANCE) for caspase-3.
AuthorsKarvinen J, Hurskainen P, Gopalakrishnan S, Burns D, Warrior U, Hemmilä I
JournalJ Biomol Screen
PubMed ID12097185
'In addition to kinases and G protein-coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. ... More
Evaluation of the phosphorescent palladium(II)-coproporphyrin labels in separation-free hybridization assays.
AuthorsBurke M, O'Sullivan PJ, Soini AE, Berney H, Papkovsky DB
JournalAnal Biochem
PubMed ID12927834
'Palladium(II)-coproporphyrin label and a set of corresponding monofunctional labeling reagents with different linker arms were evaluated for labeling of oligonucleotides and subsequent use in hybridization assays. The properties of resulting oligonucleotide probes including phosphorescence spectra, quantum yields, lifetimes, and labeling yields were examined as functions of the label and oligonucleotide ... More
A fluorescent lectin array using supramolecular hydrogel for simple detection and pattern profiling for various glycoconjugates.
AuthorsKoshi Y, Nakata E, Yamane H, Hamachi I
JournalJ Am Chem Soc
PubMed ID16895406
'Because sugar and its derivatives play important roles in various biological phenomena, the rapid and high-throughput analysis of various glycoconjugates is keenly desirable. We describe herein the construction of a novel fluorescent lectin array for saccharide detection using a supramolecular hydrogel matrix. In this array, the fluorescent lectins were noncovalently ... More
Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction.
AuthorsLiu Y, Kati W, Chen CM, Tripathi R, Molla A, Kohlbrenner W
JournalAnal Biochem
PubMed ID10036138
'A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important ... More
In situ amplification using universal energy transfer-labeled primers.
AuthorsNuovo GJ, Hohman RJ, Nardone GA, Nazarenko IA
JournalJ Histochem Cytochem
PubMed ID10026230
'We developed an amplification detection system in which a universal energy transfer-labeled primer (UniPrimer) is used in combination with any target-specific primer pair. The target specific primers each have a 5'' tail sequence, which is homologous to the 3'' end of the UniPrimer which, in turn, has a hairpin structure ... More
A real-time fluorogenic phospholipase A(2) assay for biochemical and cellular activity measurements.
'A fluorogenic analog of the PLA(2) substrate PC, named Dabcyl-BODIPY-PC, or simply DBPC, was synthesized with a fluorescence quencher (Dabcyl, 4-[(4-[N,N-dimethylamino]phenyl)azo]benzoic acid) in the sn-1 acyl chain and a BODIPY fluor in the sn-2 acyl chain. DBPC was recognized by sPLA(2) from each of the four sources examined (bee venom, ... More
A real-time assay for DNA sticky-end pairing using molecular beacons.
AuthorsLi JJ, Tan W
JournalAnal Biochem
PubMed ID12531213
Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase.
AuthorsAnderson RD, Zhou J, Hecht SM
JournalJ Am Chem Soc
PubMed ID12175203
Simultaneous detection of TaqMan probes containing Fam and Tamra reporter fluorophores.
AuthorsNasarabadi S, Milanovich F, Richards J, Belgrader P
JournalBiotechniques
PubMed ID10631486
Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria.
AuthorsSchofield P, Pell AN, Krause DO
JournalAppl Environ Microbiol
PubMed ID9055429
Molecular beacons are fluorescent probes developed for solution rather than membrane hybridization. We have investigated the utility of these probes to study rumen microbial ecology. Two cellulolytic species, Ruminococcus albus and Fibrobacter succinogenes, were tested. Membrane and solution hybridizations gave similar results in competition experiments with cocultures of R. albus ... More
Quenching of biotinylated aequorin bioluminescence by dye-labeled avidin conjugates: application to homogeneous bioluminescence resonance energy transfer assays.
AuthorsAdamczyk M, Moore JA, Shreder K
JournalOrg Lett
PubMed ID11405714
[see reaction]. Avidin conjugates containing the covalently attached dyes QSY-7 and dabcyl were prepared and shown to quench the bioluminescence of biotinylated aequorin. Quenching efficiency was shown to be dependent on both the label-to-avidin ratio and the concentration of the avidin conjugate. These properties were exploited to develop a homogeneous ... More
New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.
AuthorsGarcía-Echeverría C, Rich DH
JournalFEBS Lett
PubMed ID1551413
A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in ... More
Detection of telomerase activity utilizing energy transfer primers: comparison with gel- and ELISA-based detection.
AuthorsUehara H, Nardone G, Nazarenko I, Hohman RJ
JournalBiotechniques
PubMed ID10090999
We have developed a closed-tube format telomeric repeat amplification protocol (TRAP) assay for direct quantification of telomerase activity within the PCR vessel. The assay utilizes energy transfer (ET) primers, which emit fluorescence only upon incorporation into PCR products. This novel ET primer system (Amplifluor primers) has major advantages over existing ... More
Application of a miniature biochip using the molecular beacon probe in breast cancer gene BRCA1 detection.
AuthorsCulha M, Stokes DL, Griffin GD, Vo-Dinh T
JournalBiosens Bioelectron
PubMed ID15018955
We report for the first time the application of a biochip using the molecular beacon (MB) detection scheme. The usability of this biochip novel detection system for the analysis of the breast cancer gene BRCA1 is demonstrated using molecular beacon probes. The MB is designed for the BRCA1 gene and ... More
Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS).
AuthorsGershkovich AA, Kholodovych VV
JournalJ Biochem Biophys Methods
PubMed ID9029259
A prospective class of intramolecular fluorescence energy transfer substrates (IFETS) is described. In contrast to the known chromogenic and fluorogenic substrates that are used widely in the scientific and medical research, IFETS allow to control the enzymatic cleavage at any point of the peptide chain and thus permit simultaneous studies ... More
Molecular beacons: probes that fluoresce upon hybridization.
AuthorsTyagi S, Kramer FR
JournalNat Biotechnol
PubMed ID9630890
We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains ... More
Design of sensitive fluorogenic substrates for human cathepsin D.
AuthorsGulnik SV, Suvorov LI, Majer P, Collins J, Kane BP, Johnson DG, Erickson JW
JournalFEBS Lett
PubMed ID9280316
Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DABCYL)E-NH2, where X=cysteine, methylcysteine, ethylcysteine, tert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most ... More
Multicolor molecular beacons for allele discrimination.
AuthorsTyagi S, Bratu DP, Kramer FR
JournalNat Biotechnol
PubMed ID9447593
Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogenous solutions. When they bind to their targets they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore. We found that their hairpin conformation enables the use of a wide variety ... More
The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core.
AuthorsWang T, Dowal L, El-Maghrabi MR, Rebecchi M, Scarlata S
JournalJ Biol Chem
PubMed ID10713048
Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in ... More
Detection of small organic analytes by fluorescing molecular switches.
AuthorsFrauendorf C, Jäschke A
JournalBioorg Med Chem
PubMed ID11557338
A sensor system was developed for the determination of theophylline concentrations based on a theophylline-dependent allosteric ribozyme (Soukup, G. A.; Breaker, R. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3584) in combination with an RNA substrate which is double-labeled with a fluorophore and a quencher dye. In the presence ... More
Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media.
AuthorsBeekman B, Drijfhout JW, Bloemhoff W, Ronday HK, Tak PP, te Koppele JM
JournalFEBS Lett
PubMed ID8706864
Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synovial fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-Lys-NH2), containing the MMP cleavable Gly-Leu bond and ... More
Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.
AuthorsPennington MW, Thornberry NA
JournalPept Res
PubMed ID8012123
Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of ... More
Bordetella pertussis detection by spectrofluorometry using polymerase chain reaction (PCR) and a molecular beacon probe.
AuthorsPoddar SK, Le CT
JournalMol Cell Probes
PubMed ID11352597
Bordetella pertussis was detected by spectrofluorometry following PCR incorporating a molecular beacon probe in the reaction. A DNA fragment from the tandem repeat sequence region (IS 481) of the genome of B. pertussis was amplified in presence of the probe complementary to an internal segment of the amplified DNA fragment. ... More
Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues.
AuthorsKurth T, Grahn S, Thormann M, Ullmann D, Hofmann HJ, Jakubke HD, Hedstrom L
JournalBiochemistry
PubMed ID9708978
The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with ... More
A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases.
AuthorsCottaz S, Brasme B, Driguez H
JournalEur J Biochem
PubMed ID10951219
A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII, NIV-tetra-acetyl-chitopentaose. Two molecules, a fluorophore (5-(2-aminoethyl) amino-1-naphtalene-sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo-chitinase and chitobiosidase activities ... More
The herpesvirus protease: mechanistic studies and discovery of inhibitors of the human cytomegalovirus protease.
AuthorsFlynn DL, Becker DP, Dilworth VM, Highkin MK, Hippenmeyer PJ, Houseman KA, Levine LM, Li M, Moormann AE, Rankin A, Toth MV, Villamil CI, Wittwer AJ, Holwerda BC
JournalDrug Des Discov
PubMed ID9332827
The herpesvirus protease is a recently identified enzyme which is essential for viral replication. It is found in all herpesviruses and offers a new molecular target for therapeutic intervention. Its genomic structure has recently been described and consists of a large open reading frame which encodes a fusion protein containing ... More
A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases.
AuthorsEisenschmidt K, Lanio T, Jeltsch A, Pingoud A
JournalJ Biotechnol
PubMed ID12039534
We have developed an assay for online detection of DNA cleavage by restriction endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring ... More
A closed tube format for amplification and detection of DNA based on energy transfer.
AuthorsNazarenko IA, Bhatnagar SK, Hohman RJ
JournalNucleic Acids Res
PubMed ID9171107
A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on ... More
Glutathione S-transferase P1-1 (GSTP1-1) inhibits c-Jun N-terminal kinase (JNK1) signaling through interaction with the C terminus.
AuthorsWang T, Arifoglu P, Ronai Z, Tew KD
JournalJ Biol Chem
PubMed ID11279197
c-Jun N-terminal kinase (JNK)-mediated cell signaling pathways are regulated endogenously in part by protein-protein interactions with glutathione S-transferase P1-1 (GSTP1-1) (). Using purified recombinant proteins, combined with fluorescence resonance energy transfer technology, we have found that the C terminus of JNK is critical to the interaction with GSTP1-1. The apparent ... More
A new, sensitive fluorogenic substrate for papain based on the sequence of the cystatin inhibitory site.
AuthorsGauthier F, Moreau T, Lalmanach G, Brillard-Bourdet M, Ferrer-Di Martino M, Juliano L
JournalArch Biochem Biophys
PubMed ID8215429
We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-aminobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate ... More
Role of the gamma subunit prenyl moiety in G protein beta gamma complex interaction with phospholipase Cbeta.
AuthorsFogg VC, Azpiazu I, Linder ME, Smrcka A, Scarlata S, Gautam N
JournalJ Biol Chem
PubMed ID11546822
The G protein betagamma complex regulates a wide range of effectors, including the phospholipase Cbeta isozymes (PLCbetas). Prenyl modification of the gamma subunit is necessary for this activity. Evidence presented here supports a direct interaction between the G protein gamma subunit prenyl group and PLCbeta isozymes. A geranylgeranylated peptide corresponding ... More
Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions.
AuthorsLiu X, Farmerie W, Schuster S, Tan W
JournalAnal Biochem
PubMed ID10929808
Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to submicrometer sizes, have been developed for DNA/RNA analysis. The fluorescence-based biosensors have been applied in DNA/ RNA detection without the need for a dye-labeled target molecule or an intercalation reagent in the testing solution. Molecular beacons are hairpin-shaped oligonucleotides that report ... More
Molecular beacon polymerase chain reaction detection of Escherichia coli O157:H7 in milk.
AuthorsMcKillip JL, Drake M
JournalJ Food Prot
PubMed ID10914649
A fluorescently labeled oligonucleotide probe (molecular beacon) was applied to detect Escherichia coli O157:H7 in artificially contaminated skim milk during polymerase chain reaction (PCR) amplification of extracted DNA. The probe was designed to hybridize with a region of the slt-II gene coding for the A subunit and to fluoresce when ... More
Molecular Zipper: a fluorescent probe for real-time isothermal DNA amplification.
AuthorsYi J, Zhang W, Zhang DY
JournalNucleic Acids Res
PubMed ID16822854
Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products ... More
Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase, and protease reactions.
AuthorsSimeonov A, Bi X, Nikiforov TT
JournalAnal Biochem
PubMed ID12009695
We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides ... More
Real-time monitoring of intracellular mRNA hybridization inside single living cells.
AuthorsPerlette J, Tan W
JournalAnal Chem
PubMed ID11816586
A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at ... More
Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2.
AuthorsYan ZH, Ren KJ, Wang Y, Chen S, Brock TA, Rege AA
JournalAnal Biochem
PubMed ID12531198
Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of ... More
Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).
AuthorsKomano H, Rockwell N, Wang GT, Krafft GA, Fuller RS
JournalJ Biol Chem
PubMed ID10446224
The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular ... More
Conformational changes of the insulin receptor upon insulin binding and activation as monitored by fluorescence spectroscopy.
AuthorsLee J, Pilch PF, Shoelson SE, Scarlata SF
JournalBiochemistry
PubMed ID9054578
We have characterized the changes in intrinsic fluorescence that the insulin receptor undergoes upon ligand binding and autophosphorylation. The binding of insulin to its receptor results in an increase in the receptor's fluorescence intensity, emission energy and anisotropy. We monitored the time course of the anisotropy change, and these data, ... More
Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons.
AuthorsYang CJ, Wang L, Wu Y, Kim Y, Medley CD, Lin H, Tan W
JournalNucleic Acids Res
PubMed ID17557813
To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, ... More
Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.
AuthorsCapobianco JO, Lerner CG, Goldman RC
JournalAnal Biochem
PubMed ID1514700
A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, ... More
Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1.
AuthorsDowal L, Elliott J, Popov S, Wilkie TM, Scarlata S
JournalBiochemistry
PubMed ID11148035
Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three ... More
Transmembrane domain mediated self-assembly of major coat protein subunits from Ff bacteriophage.
AuthorsMelnyk RA, Partridge AW, Deber CM
JournalJ Mol Biol
PubMed ID11771966
The 50-residue major coat protein (MCP) of Ff bacteriophage exists as a single-spanning membrane protein in the Escherichia coli host inner membrane prior to assembly into lipid-free virions. Here, the molecular bases for the specificity and stoichiometry that govern the protein-protein interactions of MCP in the host membrane are investigated ... More
Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms.
AuthorsRockwell NC, Wang GT, Krafft GA, Fuller RS
JournalBiochemistry
PubMed ID9048578
Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases. To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates ... More
A new fluorescent quantitative polymerase chain reaction technique.
AuthorsShengqi W, Xiaohong W, Suhong C, Wei G
JournalAnal Biochem
PubMed ID12413453
To perform real-time detection of specific genes, a new complex probe has been designed and synthesized. Based on fluorescence resonance energy transfer (FRET), this complex probe is composed of a long-fluorescent reporter probe and a short-quenching probe. The 5' end of the fluorescent probe is connected to a fluorescein molecule, ... More
A fluorescence energy transfer method for analyzing protein oligomeric structure: application to phospholamban.
AuthorsLi M, Reddy LG, Bennett R, Silva ND, Jones LR, Thomas DD
JournalBiophys J
PubMed ID10233073
We have developed a method using fluorescence energy transfer (FET) to analyze protein oligomeric structure. Two populations of a protein are labeled with fluorescent donor and acceptor, respectively, then mixed at a defined donor/acceptor ratio. A theoretical simulation, assuming random mixing and association among protein subunits in a ring-shaped homo-oligomer, ... More
Genotypic analysis of Mycobacterium tuberculosis in two distinct populations using molecular beacons: implications for rapid susceptibility testing.
AuthorsPiatek AS, Telenti A, Murray MR, El-Hajj H, Jacobs WR, Kramer FR, Alland D
JournalAntimicrob Agents Chemother
PubMed ID10602730
Past genotypic studies of Mycobacterium tuberculosis may have incorrectly estimated the importance of specific drug resistance mutations due to a number of sampling biases including an overrepresentation of multidrug-resistant (MDR) isolates. An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays. ... More
Semiautomated clone verification by real-time PCR using molecular beacons.
Authorsvan Schie RC, Marras SA, Conroy JM, Nowak NJ, Catanese JJ, de Jong PJ
JournalBiotechniques
PubMed ID11126133
Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) ... More
Differential association of the pleckstrin homology domains of phospholipases C-beta 1, C-beta 2, and C-delta 1 with lipid bilayers and the beta gamma subunits of heterotrimeric G proteins.
AuthorsWang T, Pentyala S, Rebecchi MJ, Scarlata S
JournalBiochemistry
PubMed ID9931017
Pleckstrin homology (PH) domains are recognized in more than 100 different proteins, including mammalian phosphoinositide-specific phospholipase C (PLC) isozymes (isotypes beta, gamma, and delta). These structural motifs are thought to function as tethering devices linking their host proteins to membranes containing phosphoinositides or beta gamma subunits of heterotrimeric GTP binding ... More
Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease.
AuthorsNarjes F, Brunetti M, Colarusso S, Gerlach B, Koch U, Biasiol G, Fattori D, De Francesco R, Matassa VG, Steinkühler C
JournalBiochemistry
PubMed ID10677236
The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the ... More
A continuous fluorescence assay of renin activity.
AuthorsWang GT, Chung CC, Holzman TF, Krafft GA
JournalAnal Biochem
PubMed ID8512070
A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy ... More
Stains, labels and detection strategies for nucleic acids assays.
AuthorsKricka LJ
JournalAnn Clin Biochem
PubMed ID11928759
Selected developments and trends in stains, labels and strategies for detecting and measuring nucleic acids (DNA, RNA) and related molecules [e.g. oligo(deoxy)nucleotides, nucleic acid fragments and polymerase chain reaction products] are surveyed based on the literature in the final decade of the 20th century (1991-2000). During this period, important families ... More
Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2.
AuthorsRockwell NC, Fuller RS
JournalBiochemistry
PubMed ID11297433
Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2. These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity. Pre-steady-state studies have ... More
Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.
AuthorsReddy LG, Jones LR, Thomas DD
JournalBiochemistry
PubMed ID10194307
Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, ... More
Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.
AuthorsWinn-Deen ES
JournalMol Diagn
PubMed ID10089280
Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the ... More
Role of the major homology region in assembly of HIV-1 Gag.
AuthorsProvitera P, Goff A, Harenberg A, Bouamr F, Carter C, Scarlata S
JournalBiochemistry
PubMed ID11331022
The major homology region (MHR) is a highly conserved sequence in the gag gene of all retroviruses, including HIV-1. Its role in assembly is unknown, but deletion of the motif significantly impairs membrane binding and viral particle formation. To begin characterizing this defect, we have determined the contribution of this ... More
Simultaneous absolute quantification of target and control templates by real-time fluorescence reverse transcription-PCR using 4-(4'-dimethylaminophenylazo)benzoic acid as a dark quencher dye.
AuthorsKreuzer KA, Bohn A, Lupberger J, Solassol J, le Coutre P, Schmidt CA
JournalClin Chem
PubMed ID11238301
BACKGROUND: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. METHODS: For quantification of target and reference sequences, we used ... More
On the sequential determinants of calpain cleavage.
AuthorsTompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilágyi A, Bánóczi Z, Hudecz F, Friedrich P
JournalJ Biol Chem
PubMed ID14988399
The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action ... More