Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin.
AuthorsRen J, Sharpe JC, Collier RJ, London E
JournalBiochemistry
PubMed ID9893993
'The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the ... More
Topography of diphtheria toxin A chain inserted into lipid vesicles.
AuthorsHayashibara M, London E
JournalBiochemistry
PubMed ID15697244
'The membrane-inserting T domain of diphtheria toxin aids the low-pH-triggered translocation of the catalytic A chain of the toxin across endosomal membranes. To evaluate the role of the isolated A chain in translocation, the topography of isolated A chain inserted into model membrane vesicles was investigated using a mixture either ... More
Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1-TH3 as a hydrophilic tether.
AuthorsWang J, Rosconi MP, London E
JournalBiochemistry
PubMed ID16800637
'After low pH-triggered membrane insertion, the T domain of diphtheria toxin helps translocate the catalytic domain of the toxin across membranes. In this study, the hydrophilic N-terminal helices of the T domain (TH1-TH3) were studied. The conformation triggered by exposure to low pH and changes in topography upon membrane insertion ... More
Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine.
AuthorsLuedtke NW, Carmichael P, Tor Y
JournalJ Am Chem Soc
PubMed ID14531657
The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.
AuthorsBergström F, Hägglöf P, Karolin J, Ny T, Johansson LB
JournalProc Natl Acad Sci U S A
PubMed ID10535947
'The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In ... More
Size distribution of linear and helical polymers in actin solution analyzed by photon counting histogram.
AuthorsTerada N, Shimozawa T, Ishiwata S, Funatsu T
JournalBiophys J
PubMed ID17172301
'Actin is a ubiquitous protein that is a major component of the cytoskeleton, playing an important role in muscle contraction and cell motility. At steady state, actin monomers and filaments (F-actin) coexist, and actin subunits continuously attach and detach at the filament ends. However, the size distribution of actin oligomers ... More
Effects of fluorescent probe structure on the dynamics at cysteine-34 within bovine serum albumin: evidence for probe-dependent modulation of the cybotactic region.
'We have prepared a series of bovine serum albumins (BSA) that have been site-selectively labeled at cysteine-34 with one of four different sulfhydryl-selective boron dipyrromethene difluoride (BODIPY) fluorescent probes (BODIPY FL IA, BODIPY FL C(1) IA, BODIPY 530/550 IA, and BODIPY 493/503 MB). We determine how the choice of extrinsic ... More
The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted.
AuthorsHägglöf P, Bergström F, Wilczynska M, Johansson LB, Ny T
JournalJ Mol Biol
PubMed ID14687577
'Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1'' residues acting as a "bait" for cognate ... More
Donor-donor energy migration for determining intramolecular distances in proteins: I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1).
AuthorsKarolin J, Fa M, Wilczynska M, Ny T, Johansson LB
JournalBiophys J
PubMed ID9449305
'A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain ... More
Nitric oxide stress induces different responses but mediates comparable protein thiol protection in Bacillus subtilis and Staphylococcus aureus.
AuthorsHochgräfe F, Wolf C, Fuchs S, Liebeke M, Lalk M, Engelmann S, Hecker M,
JournalJ Bacteriol
PubMed ID18487332
The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO ... More
The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation.
AuthorsRamachandran R, Tweten RK, Johnson AE
JournalProc Natl Acad Sci U S A
PubMed ID15878993
FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the ... More
A fluorescent probe of polyamine transport accumulates into intracellular acidic vesicles via a two-step mechanism.
AuthorsSoulet D, Gagnon B, Rivest S, Audette M, Poulin R
JournalJ Biol Chem
PubMed ID15208319
Mammalian polyamine carriers have not yet been molecularly identified. The fluoroprobe Spd-C2-BODIPY faithfully reports polyamine transport and accumulates almost exclusively in polyamine-sequestering vesicles (PSVs). Polyamines might thus be imported first by a plasma membrane carrier and then sequestered into pre-existing PSVs (model A), or be directly captured by polyamine receptors ... More
Structural requirements for ligand binding by a probable plant vacuolar sorting receptor.
Authors Cao X; Rogers S W; Butler J; Beevers L; Rogers J C;
JournalPlant Cell
PubMed ID10760239
How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds ... More
A parallel multiharmonic frequency-domain fluorometer for measuring excited-state decay kinetics following one-, two-, or three-photon excitation.
AuthorsWatkins AN, Ingersoll CM, Baker GA, Bright FV
JournalAnal Chem
PubMed ID9726164
We report on the performance of a new, multiharmonic frequency-domain instrument that uses the high harmonic content of a passively mode-locked, pulse-picked femto-second Ti-sapphire laser as the excitation source for the determination of one-, two-, or three-photon excited time-resolved fluorescence anisotropy and intensity decay kinetics. In operation, the new instrument ... More
Dependence of M13 major coat protein oligomerization and lateral segregation on bilayer composition.
AuthorsFernandes F, Loura LM, Prieto M, Koehorst R, Spruijt RB, Hemminga MA
JournalBiophys J
PubMed ID14507706
M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded ... More
Thiol-reactive dyes for fluorescence labeling of proteomic samples.
AuthorsTyagarajan K, Pretzer E, Wiktorowicz JE
JournalElectrophoresis
PubMed ID12874870
Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA ... More
Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening.
The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these ... More
Role of endocytosis in the internalization of spermidine-C(2)-BODIPY, a highly fluorescent probe of polyamine transport.
AuthorsSoulet D, Covassin L, Kaouass M, Charest-Gaudreault R, Audette M, Poulin R
JournalBiochem J
PubMed ID12097141
The mechanism of transmembrane polyamine internalization in mammalian cells remains unknown. A novel fluorescent spermidine conjugate [Spd-C(2)-BODIPY; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-N'-(S -[spermidine-(N(4)-ethyl)]thioacetyl)ethylenediamine] was synthesized from N(4)-(mercaptoethyl)spermidine by a simple, one-step coupling procedure. In Chinese-hamster ovary (CHO) cells, Spd-C(2)-BODIPY accumulation was inhibited by exogenous putrescine, spermidine and spermine, was subject to feedback transport inhibition ... More
Fluorescent substrates of sister-P-glycoprotein (BSEP) evaluated as markers of active transport and inhibition: evidence for contingent unequal binding sites.
AuthorsWang EJ, Casciano CN, Clement RP, Johnson WW
JournalPharm Res
PubMed ID12739759
PROPOSE: Although sister-P-glycoprotein (SPGP, BSEP) is closely related to P-glycoprotein, it is much more selective in distribution and substrate recognition. Moreover, because inhibition or lack of BSEP function has severe consequences including cholestasis, hepatotoxicity, exposure to toxic xenobiotics, and drug interactions, in vitro methods are necessary for quantifying and characterizing ... More
Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width.
AuthorsWang Y, Malenbaum SE, Kachel K, Zhan H, Collier RJ, London E
JournalJ Biol Chem
PubMed ID9312118
The alpha-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes. In this report, T domain structure was studied using site-directed single-Cys mutants. The residues chosen, 322 (near the ... More
Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain.
AuthorsKachel K, Ren J, Collier RJ, London E
JournalJ Biol Chem
PubMed ID9722516
The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and ... More
Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues.
Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 '-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max ... More
Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.
AuthorsFa M, Bergström F, Karolin J, Johansson LB, Ny T
JournalEur J Biochem
PubMed ID10848991
The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, ... More
Site-directed targeting of plasminogen activator inhibitor-1 as an example for a novel approach in rational drug design.
AuthorsDe Taeye B, Compernolle G, Declerck PJ
JournalJ Biol Chem
PubMed ID14988411
As plasminogen activator inhibitor-1 (PAI-1), the physiological inhibitor of tissue-type plasminogen activator, is considered to be an important risk factor in several (patho)physiological conditions, many research activities focus on attempts to inhibit this serpin. The approach illustrated in the current study focuses on elucidating important interaction sites allowing the inhibition ... More
Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology.
AuthorsBergström F, Mikhalyov I, Hägglöf P, Wortmann R, Ny T, Johansson LB
JournalJ Am Chem Soc
PubMed ID11782171
A ground-state dimer (denoted D(I)) exhibiting a strong absorption maximum at 477 nm (epsilon = 97 000 M(-1)cm(-1)) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited D(I) was detected. A locally high concentration of BODIPY ... More