Solution conformation of a nitrobenzoxadiazole derivative of the polyene antibiotic nystatin: a FRET study.
AuthorsSilva L, Coutinho A, Fedorov A, Prieto M
JournalJ Photochem Photobiol B
PubMed ID14644562
'Nystatin is a polyene antibiotic frequently applied in the treatment of topical fungal infections. In this work, a 7-nitrobenz-2-oxa-1,3-diazole (NBD) hexanoyl amide derivative of nystatin was synthesized and its detailed photophysical characterization is presented. The average conformation of the labelled antibiotic in tetrahydrofuran, ethanol and methanol was determined by intramolecular ... More
Effects of gel material on fluorescence lifetime detection of dyes and dye-labeled DNA primers in capillary electrophoresis.
AuthorsLi L, McGown LB
JournalJ Chromatogr A
PubMed ID10360329
'Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. ... More
Lipid effects on aggregation of pulmonary surfactant protein SP-C studied by fluorescence energy transfer.
AuthorsHorowitz AD, Baatz JE, Whitsett JA
JournalBiochemistry
PubMed ID8373758
'The self-association of pulmonary surfactant protein SP-C in lipid vesicles was studied using fluorescence energy transfer. Bovine SP-C was labeled with two fluorescent probes, succinimidyl 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate and eosin isothiocyanate, on the amino terminus of the protein, producing NBD-SP-C and EITC-SP-C, respectively. The N-terminus of SP-C was relatively immobile between 20 ... More
Dual labeling of a binding protein allows for specific fluorescence detection of native protein.
AuthorsKarlström A, Nygren PA
JournalAnal Biochem
PubMed ID11476541
'Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from ... More
Synthesis and pharmacological activity of fluorescent histamine H1 receptor antagonists related to mepyramine.
AuthorsLi L, Kracht J, Peng S, Bernhardt G, Buschauer A
JournalBioorg Med Chem Lett
PubMed ID12657255
'Fluorescently labeled histamine H(1) receptor antagonists were synthesized starting from N-demethylmepyramine by introduction of omega-aminoalkyl chains (2-8 methylene groups in length) followed by derivatization of the terminal NH(2) group with various fluorophores (fluorescein, naphthofluorescein, rhodamine, tetramethylrhodamine, BODIPY, dansyl, and nitrobenzoxadiazole (NBD)). On the isolated guinea pig ileum and in a ... More
Identification of the binding surface on Cdc42Hs for p21-activated kinase.
AuthorsGuo W, Sutcliffe MJ, Cerione RA, Oswald RE
JournalBiochemistry
PubMed ID9760238
'The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has ... More
Maleimide-functionalized lipids that anchor polypeptides to lipid bilayers and membranes.
AuthorsElliott JT, Prestwich GD
JournalBioconjug Chem
PubMed ID11087332
'Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular ... More
Investigation of nonspecific effects of different dyes in the screening of labeled carbohydrates against immobilized proteins.
AuthorsDowlut M, Hall DG, Hindsgaul O
JournalJ Org Chem
PubMed ID16292809
'[structure: see text] Carbohydrates play an important role in life processes, and combinatorial chemistry can provide useful sources of thousands of synthetic carbohydrates as potential ligands for biological receptors. To accelerate the detection of positive hits arising from specific interactions between a carbohydrate and a protein, the use of fluorescent ... More
The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation.
AuthorsCrowley KS, Reinhart GD, Johnson AE
JournalCell
PubMed ID8513496
'The signal sequence is in an aqueous milieu at an early stage in the translocation of a nascent secretory protein across the endoplasmic reticulum membrane. This was determined using fluorescent probes incorporated into the signal sequence of fully assembled ribosome-nascent chain-membrane complexes: the fluorescence lifetimes revealed that the probes were ... More
Definition of the switch surface in the solution structure of Cdc42Hs.
'Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility. Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of ... More
Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.
AuthorsGlaser M, Wanaski S, Buser CA, Boguslavsky V, Rashidzada W, Morris A, Rebecchi M, Scarlata SF, Runnels LW, Prestwich GD, Chen J, Aderem A, Ahn J, McLaughlin S
JournalJ Biol Chem
PubMed ID8824266
'The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction ... More
Protein translocation across the ER membrane: a fluorescent light at the end of the tunnel.
AuthorsJohnson AE
JournalTrends Biochem Sci
PubMed ID8108855
Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore.
AuthorsCrowley KS, Liao S, Worrell VE, Reinhart GD, Johnson AE
JournalCell
PubMed ID8062388
The environment of secretory proteins undergoing translocation across the ER membrane was determined by incorporating fluorescent probes into nascent chains during translation. Dyes were positioned at various locations across the entire bilayer and inside the ribosome, and in each case the probes were in an aqueous milieu, as shown both ... More
Application of an environmentally sensitive fluorophore for rapid analysis of the binding and internalization efficiency of gene carriers.
AuthorsBergen JM, Kwon EJ, Shen TW, Pun SH,
JournalBioconjug Chem
PubMed ID18062659
Nonviral gene carriers must associate with and become internalized by cells in order to mediate efficient transfection. Methods to quantitatively measure and distinguish between cell association and internalization of delivery vectors are necessary to characterize the trafficking of vector formulations. Here, we demonstrate the utility of nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled oligonucleotides for ... More
Single giant vesicle rupture events reveal multiple mechanisms of glass-supported bilayer formation.
AuthorsHamai C, Cremer PS, Musser SM,
JournalBiophys J
PubMed ID17189305
The formation of supported lipid bilayers (SLBs) on glass from giant unilamellar vesicles (GUVs) was studied using fluorescence microscopy. We show that GUV rupture occurs by at least four mechanisms, including 1), spontaneous rupture of isolated GUVs yielding almost heart-shaped bilayer patches (asymmetric rupture); 2), spontaneous rupture of isolated GUVs ... More
Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing.
AuthorsNunnally BK, He H, Li LC, Tucker SA, McGown LB
JournalAnal Chem
PubMed ID9212706
Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of ... More
Enhancement of molecular fluorescence near the surface of colloidal metal films.
AuthorsSokolov K, Chumanov G, Cotton TM
JournalAnal Chem
PubMed ID9751028
Fluorescence enhancement was studied on silver colloidal metal films (CMFs) using two systems: (1) Langmuir--Blodgett monolayers of fluorescein-labeled phospholipids separated from the surface of the films by spacer layers of octadecanoic acid and (2) biotin--fluorescein conjugates captured by avidin molecules adsorbed on top of a multilayer structure formed by alternating ... More
Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranes.
AuthorsSilva L, Coutinho A, Fedorov A, Prieto M
JournalBiochim Biophys Acta
PubMed ID14637021
Nystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the present study, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free and ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). The mean ... More
Detection and characterization of ceramide-1-phosphate phosphatase activity in rat liver plasma membrane.
AuthorsBoudker O, Futerman AH
JournalJ Biol Chem
PubMed ID8408075
A calcium-dependent ceramide (Cer) kinase was recently detected in human leukemia (HL-60) cells (Kolesnick, R.N., and Hemer, M.R. (1990) J. Biol. Chem. 265, 18803-18808) where it may function in terminating the regulatory effects of Cer, and in synaptic vesicles (Bajjalieh, S. M., Martin, T. F. J., and Floor, E. (1989) ... More
Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.
AuthorsNomanbhoy TK, Leonard DA, Manor D, Cerione RA
JournalBiochemistry
PubMed ID8605211
The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate (sNBD) at a single reactive lysine residue. ... More
Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface.
AuthorsStevens WK, Vranken W, Goudreau N, Xiang H, Xu P, Ni F
JournalBiochemistry
PubMed ID10320322
Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a common sequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with a consensus of I-S-x-P-(x)2-4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071-29074], has been shown to be ... More
Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers.
Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis. Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids. We describe a carrier system for intracellular ... More
Synthesis and biological activity of fluorescent yeast pheromones.
AuthorsSherrill C, Khouri O, Zeman S, Roise D
JournalBiochemistry
PubMed ID7893651
The mating pheromones of Saccharomyces cerevisiae and derivatives of these pheromones have been synthesized and tested for biological activity in a solution-phase assay. The effects of native alpha-factor and a-factor on the growth of target cells in these assays were identical. A derivative of alpha-factor in which the amino terminus ... More
Alternate aggregation pathways of the Alzheimer beta-amyloid peptide. An in vitro model of preamyloid.
AuthorsHuang TH, Yang DS, Fraser PE, Chakrabartty A
JournalJ Biol Chem
PubMed ID10961999
Deposition of amyloid-beta (Abeta) aggregates in the brain is a defining characteristic of Alzheimer's disease (AD). Fibrillar amyloid, found in the cores of senile plaques, is surrounded by dystrophic neurites. In contrast, the amorphous Abeta (also called preamyloid) in diffuse plaques is not associated with neurodegeneration. Depending on the conditions, ... More
Synthetic mimics of small Mammalian cell surface receptors.
AuthorsBoonyarattanakalin S, Martin SE, Dykstra SA, Peterson BR
JournalJ Am Chem Soc
PubMed ID15600339
Receptors on the surface of mammalian cells promote the uptake of cell-impermeable ligands by receptor-mediated endocytosis. To mimic this process, we synthesized small molecules designed to project anti-dinitrophenyl antibody-binding motifs from the surface of living Jurkat lymphocytes. These synthetic receptors comprise N-alkyl derivatives of 3beta-cholesterylamine as the plasma membrane anchor ... More
Dihydroceramide biology. Structure-specific metabolism and intracellular localization.
AuthorsKok JW, Nikolova-Karakashian M, Klappe K, Alexander C, Merrill AH
JournalJ Biol Chem
PubMed ID9261117
This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis. When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl-DH-Cer (C6-NBD-DH-Cer) was incubated with HT29, NRK, BHK, or HL-60 cells, it was efficiently converted to dihydrosphingomyelin and dihydroglucosylceramide, and a number of other sphingolipids, with the ... More