Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers.
AuthorsKiuchi T, Ohashi K, Kurita S, Mizuno K
JournalJ Cell Biol
PubMed ID17470633
'Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using ... More
The citrate carrier CitS probed by single-molecule fluorescence spectroscopy.
AuthorsKästner CN, Prummer M, Sick B, Renn A, Wild UP, Dimroth P
JournalBiophys J
PubMed ID12609868
'A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were ... More
Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility.
AuthorsFischer RS, Fritz-Six KL, Fowler VM
JournalJ Cell Biol
PubMed ID12707310
'Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is ... More
Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient.
AuthorsKrantz BA, Finkelstein A, Collier RJ
JournalJ Mol Biol
PubMed ID16343527
'Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF ... More
Labeling of DNA via rearrangement of S-2-aminoethyl phosphorothioates to N-(2-mercaptoethyl)phosphoramidates.
AuthorsChen M, Gothelf KV,
JournalOrg Biomol Chem
PubMed ID18292883
The reaction of phosphorothioates in DNA with 2-bromoethylammonium bromide results in S-2-aminoethyl phosphorothioates, which can rearrange to N-(2-mercaptoethyl)phosphoramidates providing a facile method for the generation of site-specific thiol labeling of DNA sequences. The applicability of this method was demonstrated by conjugation of the thiolated DNA sequence with Na-(3-maleimidylpropionyl) biocytin and ... More
Designing biosensors for Rho family proteins--deciphering the dynamics of Rho family GTPase activation in living cells.
AuthorsPertz O, Hahn KM
JournalJ Cell Sci
PubMed ID15020671
Rho family GTPases are molecular switches that couple changes in the extracellular environment to intracellular signal transduction pathways. Their ability to regulate behaviors such as cell motility suggests very tight kinetic and spatial control of their activity, which is missed in most biochemical assays. Fluorescent probes that non-invasively report the ... More
Localized Rac activation dynamics visualized in living cells.
Signaling proteins are thought to be tightly regulated spatially and temporally in order to generate specific and localized effects. For Rac and other small guanosine triphosphatases, binding to guanosine triphosphate leads to interaction with downstream targets and regulates subcellular localization. A method called FLAIR (fluorescence activation indicator for Rho proteins) ... More
Site-specific fluorescence reveals distinct structural changes induced in the human rho 1 GABA receptor by inhibitory neurosteroids.
The rho 1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the rho 1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations ... More
Site-specific fluorescence reveals distinct structural changes with GABA receptor activation and antagonism.
AuthorsChang Y, Weiss DS
JournalNat Neurosci
PubMed ID12368804
Neurotransmitter-operated ion channels, such as the GABA (gamma-aminobutyric acid) receptor, are important in fast synaptic transmission between neurons. Using site-specific fluorescent labeling and simultaneous electrophysiological analysis in Xenopus laevis oocytes expressing recombinant rho1 GABA receptors, we identified agonist-mediated molecular rearrangements at three positions within and near the agonist-binding pocket that ... More
Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore.
AuthorsViero G, Cunaccia R, Prévost G, Werner S, Monteil H, Keller D, Joubert O, Menestrina G, Dalla Serra M
JournalBiochem J
PubMed ID16241903
Staphylococcal gamma-haemolysin HlgA-HlgB forms a beta-barrel transmembrane pore in cells and in model membranes. The pore is formed by the oligomerization of two different proteins and a still debated number of monomers. To clarify the topology of the pore, we have mutated single residues - placed near the right and ... More
Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer.
AuthorsLeifert WR, Bailey K, Cooper TH, Aloia AL, Glatz RV, McMurchie EJ
JournalAnal Biochem
PubMed ID16729956
G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit ... More
A fluorescence resonance energy transfer sensor for the beta-domain of metallothionein.
AuthorsHong SH, Maret W
JournalProc Natl Acad Sci U S A
PubMed ID12618543
We have designed a nanosensor to study the potential function of metallothionein (MT) in metal transfer and its interactions with redox partners and ligands by attaching two fluorescent probes to recombinant human MT. The specific labeling takes advantage of two different modification reactions. One is based on the fact that ... More
Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase.
AuthorsWestphal AH, Matorin A, Hink MA, Borst JW, van Berkel WJ, Visser AJ
JournalJ Biol Chem
PubMed ID16492664
We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair Förster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels ... More
Fluorometric measurements of conformational changes in glutamate transporters.
Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent extracellular glutamate concentrations from reaching neurotoxic levels (1). It is thought that glutamate transporters mediate glutamate transport through a reaction cycle with conformational changes between the two major access states that alternatively ... More
Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.
AuthorsTricerri MA, Behling Agree AK, Sanchez SA, Bronski J, Jonas A
JournalBiochemistry
PubMed ID11305923
The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids ("picket fence" model). However, based on ... More
Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions.
AuthorsKuzmenkina EV, Heyes CD, Nienhaus GU
JournalProc Natl Acad Sci U S A
PubMed ID16221762
Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of ... More
Synthesis, characterization, and application of cy-dye- and alexa-dye-labeled hongotoxin(1) analogues. The first high affinity fluorescence probes for voltage-gated K+ channels.
AuthorsPragl B, Koschak A, Trieb M, Obermair G, Kaufmann WA, Gerster U, Blanc E, Hahn C, Prinz H, Schütz G, Darbon H, Gruber HJ, Knaus HG
JournalBioconjug Chem
PubMed ID12009929
Hongotoxin(1) (HgTX(1)), a 39-residue peptide recently isolated from the venom of Centruroides limbatus, blocks the voltage-gated K+ channels K(v)1.1, K(v)1.2, and K(v)1.3 at picomolar toxin concentrations (Koschak, A., Bugianesi, R. M., Mitterdorfer, J., Kaczorowski, G. J., Garcia, M. L., and Knaus, H. G. (1998) J. Biol. Chem. 273, 2639-2644). In ... More