Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.
AuthorsPashkova A, Chen HS, Rejtar T, Zang X, Giese R, Andreev V, Moskovets E, Karger BL
JournalAnal Chem
PubMed ID15801742
The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of ... More
Micron-scale spatially patterned, covalently immobilized vascular endothelial growth factor on hydrogels accelerates endothelial tubulogenesis and increases cellular angiogenic responses.
AuthorsLeslie-Barbick JE, Shen C, Chen C, West JL,
JournalTissue Eng Part A
PubMed ID20712418
'Spontaneous formation of endothelial tubules was restricted to patterned micron-scale regions presenting cell adhesion ligands and angiogenic signaling protein on poly(ethylene glycol) hydrogels. Arginine-glycine-aspartic acid-serine (RGDS), an integrin ligand, and vascular endothelial growth factor (VEGF), a rate-limiting signaling protein involved in angiogenesis, were covalently bound through photopolymerization via laser scanning ... More
Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate.
AuthorsMartin K, Hart C, Schulenberg B, Jones L, Patton WF
JournalProteomics
PubMed ID11987124
'A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR-X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39-phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all ... More
Polar localization of virulence-related Esx-1 secretion in mycobacteria.
AuthorsCarlsson F, Joshi SA, Rangell L, Brown EJ,
JournalPLoS Pathog
PubMed ID19180234
'The Esx-1 (type VII) secretion system is critical for virulence of both Mycobacterium tuberculosis and Mycobacterium marinum, and is highly conserved between the two species. Despite its importance, there has been no direct visualization of Esx-1 secretion until now. In M. marinum, we show that secretion of Mh3864, a novel ... More
Pulsed fluorescence measurements of trapped molecular ions with zero background detection.
AuthorsKhoury JT, Rodriguez-Cruz SE, Parks JH
JournalJ Am Soc Mass Spectrom
PubMed ID12056569
'Sensitive methods have been developed to measure laser-induced fluorescence from trapped ions by reducing the detection of background scattering to zero levels during the laser excitation pulse. The laser beam diameter has been reduced to approximately 150 microm to eliminate scattering on trap apertures and the resulting laser-ion interaction is ... More
Analysis of proteins stained by Alexa dyes.
AuthorsHuang S, Wang H, Carroll CA, Hayes SJ, Weintraub ST, Serwer P
JournalElectrophoresis
PubMed ID15004835
'Alexa dye staining of proteins is used for the fluorescence microscopy of single particles that are sometimes multimolecular protein complexes. To characterize the staining, post-staining determination must be made of which protein(s) in a complex have been Alexa-stained. The present communication describes the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis ... More
Chemoselective ligation in the functionalization of polysaccharide-based particles.
'Despite the promise of precisely targeted or otherwise functionalized polymeric particulate drug delivery vehicles, typical biocompatible particles are generally not amenable to facile and selective surface modification. Herein, we report the development of a simple, mild, and chemoselective strategy for the conjugation of biologically active molecules to the surface of ... More
Small heat shock protein Hsp16.3 modulates its chaperone activity by adjusting the rate of oligomeric dissociation.
AuthorsFu X, Liu C, Liu Y, Feng X, Gu L, Chen X, Chang Z
JournalBiochem Biophys Res Commun
PubMed ID14521926
'Small heat shock proteins usually exist as oligomers and appear to undergo dynamic dissociation/reassociation, with oligomeric dissociation being a prerequisite for their chaperone activities. However, contradictory cases were also reported that chaperone activities could be enhanced with no change or even increase in oligomeric sizes. Using Hsp16.3 as a model ... More
Distribution of the surfactant-associated protein C within a lung surfactant model film investigated by near-field optical microscopy.
AuthorsKramer A, Wintergalen A, Sieber M, Galla HJ, Amrein M, Guckenberger R
JournalBiophys J
PubMed ID10620309
'Lung surfactant films at the air/water interface exhibit the particularity that surfactant molecules are expelled from the surface monolayer into a surface associated multilamellar phase during compression. They are able to re-enter the surface film during the following expansion. The underlying mechanism for this behavior is not fully understood yet. ... More
Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis.
AuthorsBernardo J, Long HJ, Simons ER,
JournalCytometry A
PubMed ID19937952
'Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within ... More
FRET-based mapping of calmodulin bound to the RyR1 Ca2+ release channel.
AuthorsCornea RL, Nitu F, Gruber S, Kohler K, Satzer M, Thomas DD, Fruen BR,
JournalProc Natl Acad Sci U S A
PubMed ID19332786
'Calmodulin (CaM) functions as a regulatory subunit of ryanodine receptor (RyR) channels, modulating channel activity in response to changing [Ca(2+)](i). To investigate the structural basis of CaM regulation of the RyR1 isoform, we used site-directed labeling of channel regulatory subunits and fluorescence resonance energy transfer (FRET). Donor fluorophore was targeted ... More
Characterization of alpha-crystallin-plasma membrane binding.
AuthorsCobb BA, Petrash JM
JournalJ Biol Chem
PubMed ID10692476
'Alpha-crystallin, a large lenticular protein complex made up of two related subunits (alphaA- and alphaB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and ... More
Fluorescent labeling of cell-free synthesized proteins by incorporation of fluorophore-conjugated nonnatural amino acids.
AuthorsKang SH, Jun SY, Kim DM
JournalAnal Biochem
PubMed ID17113028
'Although fluorescent dyes, such as fluorescein derivatives, have bulky and complex structures, nonnatural amino acids carrying these fluorescein derivatives are acceptable by the Escherichia coli ribosome and are useful for the cotranslational fluorescent labeling of cell-free synthesized proteins. Surprisingly, the incorporation efficiency of nonnatural amino acids carrying fluorescein derivatives into ... More
Porous functionalised silica particles: a potential platform for biomolecular screening.
AuthorsJohnston AP, Battersby BJ, Lawrie GA, Trau M,
JournalChem Commun (Camb)
PubMed ID15700057
Novel, porous, functionalised silica particles have been developed with controlled morphology, which promote covalent attachment of fluorescent dyes which can act as an optical barcode.
Design of high-affinity peptide conjugates with optimized fluorescence quantum yield as markers for small peptide transporter PEPT1 (SLC15A1).
AuthorsBahadduri PM, Ray A, Khandelwal A, Swaan PW,
JournalBioorg Med Chem Lett
PubMed ID18374571
We employed a computational approach to design and synthesize a series of fluorescently labeled hPEPT1 substrates. Five Alexa Fluor-350-labeled peptides were assessed for their in vitro inhibitory activity in hPEPT1-transfected CHO cells. At least four labeled peptides show potent inhibitory activity toward hPEPT1-mediated uptake of [(3)H]-GlySar and three compounds displayed ... More
Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.
AuthorsFalati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B
JournalNat Med
PubMed ID12244306
We have used confocal and widefield microscopy to image thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and of a brightfield channel. Vascular injury is induced with a laser focused through the microscope optics. ... More
Arf6 coordinates actin assembly through the WAVE complex, a mechanism usurped by Salmonella to invade host cells.
AuthorsHumphreys D, Davidson AC, Hume PJ, Makin LE, Koronakis V,
Journal
PubMed ID24085844
ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We ... More
Sometimes it takes two to tango: contributions of dimerization to functions of human a-defensin HNP1 peptide.
AuthorsPazgier M, Wei G, Ericksen B, Jung G, Wu Z, de Leeuw E, Yuan W, Szmacinski H, Lu WY, Lubkowski J, Lehrer RI, Lu W,
JournalJ Biol Chem
PubMed ID22270360
Human myeloid a-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the ... More
High constitutive activity is an intrinsic feature of ghrelin receptor protein: a study with a functional monomeric GHS-R1a receptor reconstituted in lipid discs.
AuthorsDamian M, Marie J, Leyris JP, Fehrentz JA, Verdié P, Martinez J, Banères JL, Mary S,
JournalJ Biol Chem
PubMed ID22117076
Despite its central role in signaling and the potential therapeutic applications of inverse agonists, the molecular mechanisms underlying G protein-coupled receptor (GPCR) constitutive activity remain largely to be explored. In this context, ghrelin receptor GHS-R1a is a peculiar receptor in the sense that it displays a strikingly high, physiologically relevant, ... More
Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+-ATPase to inhibit phagosome acidification.
AuthorsWong D, Bach H, Sun J, Hmama Z, Av-Gay Y,
JournalProc Natl Acad Sci U S A
PubMed ID22087003
Mycobacterium tuberculosis (Mtb) pathogenicity depends on its ability to inhibit phagosome acidification and maturation processes after engulfment by macrophages. Here, we show that the secreted Mtb protein tyrosine phosphatase (PtpA) binds to subunit H of the macrophage vacuolar-H(+)-ATPase (V-ATPase) machinery, a multisubunit protein complex in the phagosome membrane that drives ... More
Fluorescence spectroscopic profiling of compound libraries.
AuthorsSimeonov A, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall NT, Shinn P, Smith J, Austin CP, Auld DS, Inglese J,
JournalJ Med Chem
PubMed ID18363325
Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions ... More
Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.
AuthorsCox WG, Singer VL,
JournalBiotechniques
PubMed ID14740493
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized ... More
Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells.
AuthorsShalek AK, Robinson JT, Karp ES, Lee JS, Ahn DR, Yoon MH, Sutton A, Jorgolli M, Gertner RS, Gujral TS, Macbeath G, Yang EG, Park H,
JournalProc Natl Acad Sci U S A
PubMed ID20080678
A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies ... More
An affinity-based method for the purification of fluorescently-labeled biomolecules.
AuthorsNguyen T, Joshi NS, Francis MB
JournalBioconjug Chem
PubMed ID16848391
Due to the difficulty of separating mixtures of labeled and unlabeled biomolecules, a general new method for the affinity purification of modified proteins has been developed. A Sepharose-based solid support bearing beta-cyclodextrin groups was used to capture chromophore-modified proteins selectively, while unmodified proteins remained in solution. After isolation of the ... More
Kinetic preference for oriented DNA binding by the yeast TATA-binding protein TBP.
AuthorsLiu Y, Schepartz A
JournalBiochemistry
PubMed ID11371187
In solution, the TATA box binding protein from S. cerevisiae (yTBP) is only minimally oriented when bound to the adenovirus major late promoter (AdMLP) and the yeast CYC1 promoter. At equilibrium, approximately 60% of the complexes are assembled in the orientation observed within crystal structures; 40% are assembled in the ... More
Intracellular Helicobacter pylori in gastric epithelial progenitors.
AuthorsOh JD, Karam SM, Gordon JI
JournalProc Natl Acad Sci U S A
PubMed ID15795379
Helicobacter pylori is generally viewed as an extracellular pathogen. We have analyzed the tropism of H. pylori clinical isolates in a gnotobiotic transgenic mouse model of human chronic atrophic gastritis, a preneoplastic condition. These mice lack acid-producing parietal cells and have an amplified population of dividing gastric epithelial progenitors (GEPs) ... More
Poly(vinyl alcohol)-coated microfluidic devices for high-performance microchip electrophoresis.
AuthorsBelder D, Deege A, Kohler F, Ludwig M
JournalElectrophoresis
PubMed ID12412126
The channels of microfluidic glass chips have been coated with poly(vinyl alcohol) (PVA). Applied for microchip electrophoresis, the coated devices exhibited a suppressed electroosmotic flow and improved separation performance. The superior performance of PVA-coated channels could be demonstrated by electrophoretic separations of labeled amines and by video microscopy. While a ... More
Quenched auto-ligating DNAs: multicolor identification of nucleic acids at single nucleotide resolution.
AuthorsSando S, Abe H, Kool ET
JournalJ Am Chem Soc
PubMed ID14746476
We describe the synthesis and study of multicolor quenched autoligating (QUAL) probes for identification and discrimination of closely related RNA and DNA sequences in solution and in bacteria. In these probes, a dabsyl quencher doubles as an activator in the oligonucleotide-joining reaction. The oligonucleotides remain dark until they bind at ... 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
Lipid microdomain clustering induces a redistribution of antigen recognition and adhesion molecules on human T lymphocytes.
AuthorsMitchell JS, Kanca O, McIntyre BW
JournalJ Immunol
PubMed ID11884440
The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with ... More
Structural and functional changes in the alpha A-crystallin R116C mutant in hereditary cataracts.
AuthorsCobb BA, Petrash JM
JournalBiochemistry
PubMed ID11123904
alpha-Crystallin, the major protein component of vertebrate lenses, forms a large complex comprised of two homologous subunits, alphaA- and alphaB-crystallin. It has the ability to suppress stress-induced protein aggregation in vitro, bind saturably to lens plasma membranes, and aid in light refraction through short-range ordering. Recently, a missense mutation in ... More
Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin.
AuthorsBiswas A, Goshe J, Miller A, Santhoshkumar P, Luckey C, Bhat MB, Nagaraj RH
JournalBiochemistry
PubMed ID17260942
Human alphaB-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide (54FLRAPSWF61) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alphaA-crystallin. To determine whether the ... More
Screening for aneuploidies of ten different chromosomes in two rounds of FISH: a short and reliable protocol.
AuthorsBaart EB, Martini E, Van Opstal D
JournalPrenat Diagn
PubMed ID15614916
OBJECTIVE: To develop a DNA labelling protocol for the simultaneous detection of five different fluorescent chromosomal DNA probes within one round of hybridisation. In combination with a commercial five-colour probemix for the second round of hybridisation, this results in a fast and reliable Fluorescence in situ Hybridization (FISH) protocol, enabling ... More