Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “FRET”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 901 records · Page 50Linked to original sources
PubMed2005

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting.

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Algorithms↗
PubMed2003

On-line monitoring of apoptosis in insulin-secreting cells.

Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells.

Animals↗
PubMed1983

XPS analysis of 316 LVM corroded in serum and saline.

Surface chemical analysis by x-ray photoelectron spectroscopy (XPS) was undertaken on 316 LVM stainless steel in the attempt to better understand corrosion occurring in vivo. Samples were dipped in saline or in blood serum, corroded in serum or saline by the application of a 5 volt anodic potential, or corroded by fretting. The products produced by fretting corrosion were also examined. XPS analysis revealed rapid protein coating of the stainless steel surfaces exposed to serum, changes in the oxidation state of the surfaces, and changes in the chlorine on the surface. In addition it was demonstrated that the corrosion products generated by fretting in saline had an oxidation state similar to that of chronic chloride whereas the corrosion products generated in serum had an oxidation state similar to that of potassium dichromate. These findings may have important implications since the chromium in dichromate is more biologically active than that in chronic chloride.

Blood↗
PubMed2006

Selective localization of recognition complexes for leukotriene B4 and formyl-Met-Leu-Phe within lipid raft microdomains of human polymorphonuclear neutrophils.

Neutrophilic polymorphonuclear leukocytes contain glycosphingolipid- and cholesterol-enriched lipid raft microdomains within the plasma membrane. Although there is evidence that lipid rafts function as signaling platforms for CXCR chemokine receptors, their role in recognition systems for other chemotaxins such as leukotriene B4 (LTB4) and fMLP is unknown. To address this question, human neutrophils were extracted with 1% Brij-58 and fractionated on sucrose gradients. B leukotriene receptor-1 (BLT-1), the primary LTB4 receptor, partitioned to low density fractions, co-isolating with the lipid raft marker, flotillin-1. By contrast, formyl peptide receptor (FPR), the primary fMLP receptor, partitioned to high density fractions, co-isolating with a non-raft marker, Cdc42. This pattern was preserved after the cells were stimulated with LTB4 or fMLP. Fluorescence resonance energy transfer (FRET) was performed to confirm the proximity of BLT-1 and FPR with these markers. FRET was detected between BLT1 and flotillin-1 but not Cdc42, whereas FRET was detected between FPR and Cdc42, but not flotillin-1. Pretreating neutrophils with methyl-beta-cyclodextrin, a lipid raft-disrupting agent, suppressed intracellular Ca(2+) mobilization and ERK1/2 phosphorylation in response to LTB4 but had no effect on either of these responses to fMLP. We conclude that BLT-1 is physically located within lipid raft microdomains of human neutrophils and that disrupting lipid raft integrity suppresses LTB4-induced activation. By contrast, FPR is not associated with lipid rafts, and fMLP-induced signaling does not require lipid raft integrity. These findings highlight the complexity of chemotaxin signaling pathways and offer one mechanism by which neutrophils may spatially organize chemotaxin signaling within the plasma membrane.

Calcium↗
PubMed1999

High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).

Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible.

Carrier Proteins↗
PubMed2001

Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: role of the intracellular adapter protein Fe65.

Amyloid-beta, the major constituent of senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolysis. Kunitz protease inhibitor (KPI) containing forms of APP (APP751/770) interact with a multifunctional endocytic receptor, the low-density lipoprotein receptor-related protein (LRP), which modulates its proteolytic processing affecting production of amyloid-beta. We used fluorescence resonance energy transfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcellular localization and the molecular domains involved in the APP-LRP interaction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), suggesting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellular domain probes), and this ectodomain interaction is not altered by RAP. By using C-terminally tagged LRP and APP, we demonstrate a second site of interaction between the C termini of both APP695 and APP770 and the C terminus of LRP, and that the interactions at these regions are not sensitive to RAP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasmic tails of LRP and APP. FRET studies confirmed a close proximity between the amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic domain and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both extracellular and intracellular protein interaction domains.

Amyloid beta-Protein Precursor↗
PubMed2002

Hepatitis C genotype determination by melting curve analysis with a single set of fluorescence resonance energy transfer probes.

BACKGROUND: The genotype of hepatitis C virus (HCV) is a predictor of antiviral therapeutic response. We describe an approach for HCV genotype determination by real-time PCR and melting curve analysis. METHODS: After automated nucleic acid extraction, we used reverse transcription-PCR in a block cycler to amplify nucleotides 6-329 of the 5'-untranslated region of HCV. The product was further amplified by single-tube real-time seminested PCR in a LightCycler instrument (Roche). The final product was analyzed by melting curves with the use of fluorescence resonance energy transfer (FRET) probes. The FRET sensor probe was directed at nucleotides 151-170 of type 1 HCV and was designed to distinguish types 1a/b, 2a/c, 2b, 3a, and 4, with melting temperatures (T(m)s) predicted to differ by 1 degrees C. Genotypes were compared in a blinded fashion with those of the INNO-LiPA(TM) test (Bayer Diagnostics) on 111 serum samples. RESULTS: In preliminary experiments, the Mg(2+) concentration was found to be critical in allowing clear separation of melting points, with the best separation at a Mg(2+) concentration of 2 mmol/L. The results for 111 samples clustered at expected T(m)s for genotypes 1a/b (n = 78), 2a/c (n = 2), 2b (n = 11), 3a (n = 14), and 4 (n = 2). Of the 111 samples, results for 110 were concordant with the comparison method at the level of type 1, 2, 3, or 4. Subtyping results were discordant for two samples, both of type 2. For 108 samples concordant with INNO-LiPA at the genotype and subtype levels, the mean T(m)s were 64.1, 59.5, 54.2, 52.6, and 50.1 degrees C for types 1a/b, 2a/c, 4, 2b, and 3a, respectively, with SDs of 0.2, 0.3, 0.3, 0.2, and 0.3 degrees C. All 78 samples identified as type 1 were concordant with results of the comparison method. CONCLUSIONS: Melting analysis with a single pair of FRET probes can rapidly provide information about HCV genotypes and identifies type 1 samples with high specificity.

DNA Probes↗
PubMed2006

[Analysis of Toll-like receptor 4 and myeloid differentiation protein-2 interaction with fluorescence resonance energy transfer].

OBJECTIVE: To study the interaction between Toll-like receptor (TLR) 4 and myeloid differentiation protein-2 (MD-2) in living cells using fluorescence resonance energy transfer (FRET) technology. METHODS: The coding sequences of TLR4 and MD-2 (without the signal peptide sequence) were amplified by PCR and cloned into enhanced cyan fluorescence protein (CFP) and enhanced yellow fluorescence protein (YFP) expression vectors carrying TLR4 signal peptides (pECFP-C1-SP and pEYFP-C1-SP). HEK293 cells were transfected respectively or together with the reconstructed plasmids verified by enzyme digestion and sequence analysis, and the expression and sublocalization of these fluorescence proteins in the cells were observed using fluorescence microscope. FRET in the cells coexpressing CFP-TLR4 and YFP-MD-2 was detected using routine and acceptor photobleaching method. RESULTS: The reconstructed plasmids were expressed in HEK293 cells. The cyan or yellow fluorescence was located in the cytoplasm, mainly around the nucleus in the cells transfected with pECFP/TLR4 or pEYFP/MD-2, and both the cyan and yellow fluorescence located mainly in the membrane and occasional in the cytoplasm of cells cotransfected with pECFP/TLR4 and pEYFP/MD-2. Routine or acceptor photobleaching detected FRET phenomena in cells coexpressing CFP-TLR4 and YFP-MD-2, suggesting direct interaction between TLR4 and MD-2. CONCLUSION: This study provides direct evidence of the interaction between TLR4 and MD-2 in living cells.

Cell Line↗
PubMed2004

Antisense molecular beacon strategy for in situ visualization of snRNA and fibrillarin protein interaction in Giardia lamblia.

Use of confocal microscopy has provided many recent developments in the study of functional aspects, especially localization and distribution of proteins, DNA and RNA within the cells. In the present investigation, we have applied for the first time, antisense molecular beacon based Fluorescence Resonance Energy Transfer (FRET) and Flow Cytometric Energy Transfer (FCET) techniques to demonstrate binding and co-localization of fibrillarin protein with small nuclear RNA (snRNA) to form ribonucleoprotein particle (RNPP) complex in Giardia lamblia. It has been observed by FRET and FCET that energy transfer occurs from fluorescence tagged fibrillarin to snRNA antisense molecular beacon confirming the clear physical interaction between them during RNPP complex formation. This is the first demonstration of in situ detection of RNA-protein complex formation by antisense molecular beacon based FRET and FCET in Giardia lamblia.

Animals↗
PubMed1990

[Relationship between years of methamphetamine use and symptoms of methamphetamine psychosis].

The authors researched the demographic characteristics of 233 patients with methamphetamine-associated disorders, and the relation between years of methamphetamine use and symptoms of methamphetamine psychosis. The results were as follows: There were more male users than females. However there were signs that the female users were gradually increasing. Users tended to be older, but users in their 20's and 30's continued to be predominant. Their school careers were usually limited and most of them had left school at a young age. Relations with a particular social group (e.g. organized gangs) has given most of them a chance to use methamphetamine. The symptoms that were seen with high frequency at the first examination were anxiety, fretfulness, auditory hallucination, insomnia, irritability, psychomotor excitement, delusion of persecution, suspicion, delusion of reference, mistake of circumstance, loss of appetite, affective disorder, hypobulia and personality change. With these symptoms, there is a possibility that five years of methamphetamine use is the turning point in terms of the frequency of symptoms occurrence. It was suggested that affective and perceptual disorders depend on the dose of methamphetamine, but abnormalities in thought subject may be deeply influenced by the patient's "feeling of social wrong". Emotional exhilaration and euphoria decreased as the number of years of methamphetamine use increased. These phenomena may be an indication of tolerance. The symptoms that were seen with high frequency at the last examination were hypobulia, affective disorder, personality change, insomnia, anxiety and fretfulness. The symptoms that were highly resistant to treatment were hypobulia, affective disorder, personality change, general malaise, hypochondriasis, insomnia, anxiety and fretfulness. It was suggested that five years of methamphetamine use may be a turning point in the residual rate of symptoms at the last examination after treatment, and also the resistance rate to treatment. Hypobulia and personality change became more evident during treatment.

Adolescent↗
PubMed1994

Characterization of wear in composite material orthopaedic implants. Part II: The implant/bone interface.

Carbon fiber/PEEK polymer (C/PEEK) composite materials are being developed for use as orthopaedic implant materials. Wear is an issue of increasing importance in orthopaedic implants; particulate debris generated by the wearing of biomaterials may be a causal factor leading to osteolysis and implant loosening. Therefore, numerical and experimental studies were completed to characterize the wear of C/PEEK composite materials in comparison to current orthopaedic implant materials. Finite element analyses (FEA) of a composite material hip stem implanted in a femur and loaded at 890 N determined that peak contact stresses will occur at the proximal-medial and distal regions of the implant. These contact stresses were found to be below 1.0 MPa over most of the implant surface; however the peak stress in the proximal-medial region was 1.8 MPa and higher still at the distal portion of the stem. In vivo forces result in contact stress values up to 9.0 MPa. The composite implant exhibited 10-40% lower contact stresses in the distal region compared to a titanium-alloy implant of identical design. Composite material wear samples were slid against porous hydroxylapatite (HA) to simulate the stem/bone interface. An identical series of experiments was run for comparison to a current orthopaedic implant material--Ti6A14V titanium alloy. Two domains of motion were studied; a composite ring-on-HA disc large amplitude sliding wear test; and a composite pin-on-HA disc small amplitude fretting regimen. Nominal contact pressures during testing were 1.4 MPa and 7.6 MPa for sliding and fretting tests, respectively. Fretting and sliding abrasive wear tests resulted in the composite material exhibiting a lower wear rate than the titanium-alloy. The magnitude of the difference was greatly dependent on the contact pressures, sliding amplitudes, and counterface material properties.

Alloys↗
PubMed1997

Occurrence of DNA sequence differences in C-heterochromatin of Eulemur coronatus and Eulemur macaco, as revealed by fluorescence resonance energy transfer.

In the genus Eulemur (Malagasy lemurs) karyotype diversification has occurred mainly through Robertsonian mechanisms of chromosome fusion (Rumpler et al., 1976). Eulemur coronatus is the sole species to have the largest genome size, due to a very large amount of C-heterochromatin, mostly located at the pericentromeric regions of the largest chromosomes (Warter and Rumpler, 1985). This increase in C-heterochromatin was thought to be due to DNA amplification (Ronchetti et al., 1993). The aim of this work was to investigate whether the large C-heterochromatin of Eulemur coronatus might have derived by amplification of the smaller C-heterochromatin of Eulemur macaco, a closely related species with smaller genome size. To obtain information on the overall base composition of the total genomes, on the relative interspersion of AT and GC base paris along the DNA molecule and on the structural differences in C-heterochromatin, we used a quantitative cyto-chemical approach, based on fluorescence resonance energy transfer (FRET) between DNA-specific fluorochromes (i.e. the AT-specific Hoechst 33258, and the non base-specific dye, propidium iodide). Micro-spectrofluorometry and image analysis were used to investigate both the overall FRET efficiency and its spatial distribution along the chromosome arms. FRET efficiency values of the DNA in C-heterochromatin were significantly different in the two Eulemur species, indicating a different qualitative composition of repetitive DNA. This suggests that the repetitive DNA of Eulemur coronatus cannot have originated by amplification in toto of the repetitive DNA sequences of Eulemur macaco.

Animals↗
PubMed2000

Metal ion release from titanium with active oxygen species generated by rat macrophages in vitro.

The release of metal ions due to active oxygen species generated by macrophages (Mphi) phagocytosing high-density polyethylene (HDPE) particles was studied in vitro to investigate the mechanism behind the release of metal ions from titanium implants into nearby tissues in the absence of wear and fretting in vivo. To determine the effects of Mphis on metal ion release, titanium disks were immersed in different solutions and the titanium ions released from the titanium disks into each solution were quantified. The results revealed that active oxygen species generated by Mphis induced the metal ion release. In particular, the ion release was accelerated with HDPE because the Mphis that phagocytosed HDPE generated more active oxygen species than Mphis that did not phagocytose any HDPE. Metal ions were also released by organic species in the absence of Mphis. These are some of the causes for metal ion release from titanium implants in the absence of wear and fretting in vivo.

Animals↗
PubMedDate not supplied

Analysis of the effect of a peptide sequence of the E2 protein (HGV/GBV-C) on the physicochemical properties of zwitterionic and negatively charged bilayers.

The membrane-interacting properties of a potential epitope of GB virus C/hepatitis G virus located at the region (99-118) of the E2 structural protein were investigated using several fluorescence techniques. SUV of DMPC:DPPC (1:1) or DMPG:DPPC (1:1) zwitterionic and anionic mixtures, respectively, were used as model membranes. FRET with NBD-PE as energy donor and Rho-PE as energy acceptor-labelled SUV indicated that the peptide was able to fuse both zwitterionic and anionic SUVs, the latter requiring lower peptide concentrations. However, the peptide increased the steady-state anisotropy of DPH embedded in the hydrophobic centre of the membrane with zwitterionic headgroups and to a lesser extent in anionic bilayers, suggesting that charge-charge interactions are not required for membrane interactions and also confirming the FRET results. No changes in anisotropy were observed with the probe TMA-DPH located at the surface of the bilayer. Finally, analysis of the intrinsic emission fluorescence of the tryptophan residue, upon incubation with SUV, showed a blue shift in the presence of anionic bilayers, both below and above the main transition temperature (T(m)) (gel to liquid-crystalline state) and, to a lesser extent, with the zwitterionic model membrane.

Anisotropy↗
PubMedDate not supplied

Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge.

In this study, 573 nm quantum dots (QDs)-rabbit IgG-goat anti-rabbit IgG-638 nm QDs immunocomplexes were prepared, utilizing antigen-antibody interaction. 573 nm-emitting QDs were conjugated to antigen (rabbit IgG) and 638 nm-emitting QDs were conjugated to antibody (goat anti-rabbit IgG) via electrostatic/hydrophilic self-assembly, respectively. The mutual affinity of the antigen and antibody brought two kinds of QDs close enough to result in fluorescence resonance energy transfer (FRET) between them; the luminescence emission of 573 nm QDs was quenched, while that of 638 nm QDs was enhanced. The luminescence emission of 573 nm QDs could be recovered when the immunocomplexes were exposed to the unlabelled rabbit IgG antigen. The FRET efficiency (E) and the distance between the donor and the acceptor were calculated.

Absorption↗
PubMedDate not supplied

Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes.

Time-resolved Förster resonance energy transfer (trFRET) has been used to obtain interdye distance distributions. These distributions give the most probable distance as well as a parameter, sigma, that characterize the width of the distribution. This latter parameter contains information not only on the flexibility of the dyes tethered to macromolecules, but on the flexibility of the macromolecules. Both the most probable interdye distance as well as sigma provide insight into DNA static bending and DNA flexibility. Time-resolved fluorescence anisotropy and static anisotropy measurements can be combined to provide a measure of the cone angle within which the tethered dyes appear to wobble. When this motion is an order of magnitude faster than the average lifetime that characterizes transfer, an average value of the dipolar orientational parameter kappa2 can be calculated for various mutual dye orientations. The resulting kappa2 distribution is very much narrower than the limiting values of 0 and 4, allowing more precise distances and distance changes to be determined. Static and time-resolved fluorescence data can be combined to constrain the analyses of DNA-protein kinetics to provide thermodynamic parameters for binding and for conformational changes along a reaction coordinate. The parameter sigma can be used to model multiple DNA-protein complexes with varying DNA bend angles in a global fitting of trFRET data. Such a global fitting approach has shown how the range of bends in single base DNA variants, when bound by the TATA binding protein (TBP), can be understood in terms of two limiting forms. Time-resolved FRET, combined with steady-state FRET, can be used to show not only how osmolytes affect the binding of DNA to proteins, but also how DNA bending depends on osmolyte concentration in the DNA-protein complexes.

DNA↗
PubMed2002

The one-bead two-compound assay for solid phase screening of combinatorial libraries.

Fluorescent quenched substrate libraries are a very powerful tool for investigation of protease activity and specificity. Particularly, libraries where the fluorescent resonance energy transfer (FRET) pair is 3-nitrotyrosine and 2-amino-benzamide are easy to prepare by split and combine synthesis to yield a one-bead one-compound library format. The solid support is critical for the successful hydrolysis of the resin-bound substrates. For this purpose, a range of highly porous poly(ethylene glycol) (PEG)-based resins have been developed. Active substrates yield highly fluorescent beads and these are selected under a fluorescence microscope or isolated on a bead sorter. Edman sequence analysis yields the substrate sequence, the cleavage point, and the degree of conversion. The method gives a complete map of the substrate specificity, and substrates with high affinity for the active site can be selected. These may in turn be used as inhibition indicators in a second solid phase library assay for enzyme inhibition where each single bead is transformed into an assay container. The substrate is attached to temporarily shielded functional groups after completion of inhibitor library synthesis. By using a photolabile linker and ladder synthesis, the active inhibitors may be rapidly identified by mass spectrometry. In each bead, the putative inhibitor competes with the substrate attached for binding to the enzyme, and when the inhibitor binds strongly, the substrate remains intact and quenched. Thus dark beads indicate inhibitors, and these may be isolated using a bead-sorter and the structure determined by mass spectrometry. A selection of the best substrates and inhibitors should always be resynthesized for solution kinetics and confirmation of the results obtained on solid support. The inhibitor assay is almost free from false positives, which is a consequence of combining the binding of the protease to the inhibitor with observation of activity toward a FRET substrate. The K(i) of the identified inhibitors are typically in the nM range.

Amino Acid Sequence↗
PubMed2003

Reflections on apparent DNA bending by charge variants of bZIP proteins.

Basic-leucine zipper (bZIP) proteins have been studied intensely as transcription factors. It has been proposed that the bZIP domain might modulate transcription activation through the induction of conformational changes in the DNA binding site. We have been interested in using bZIP peptides as convenient models with which to study the role of asymmetric phosphate neutralization in DNA bending. DNA bending experiments have yielded discordant results for bZIP peptides studied by electrophoretic- vs solution-based assays. We review the history of DNA bending assays involving bZIP peptides and introduce the reader to examples of discordant results. Our recent published experiments designed to clarify this field of study will then be reviewed. The engineering of protein fusions has established that electrophoretic phasing assays are relatively insensitive to precise protein structure/conformation and instead appear to report DNA bending, as influenced by protein charge. New applications of time-resolved fluorescence resonance energy transfer (FRET) have allowed for the first time corroboration of electrophoretic phasing assays with solution-based FRET measurements. We report that two conventional DNA bending assays that rely on DNA ligation cannot be applied to analysis of the bZIP peptides we studied due to ligation inhibition.

Amino Acid Sequence↗