Nuclear import of the ran exchange factor, RCC1, is mediated by at least two distinct mechanisms.
AuthorsNemergut ME, Macara IG
JournalJ Cell Biol
PubMed ID10811825
'RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an approximately 45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 ... More
Evidence that translocation of the proteinase precedes its acylation in the serpin inhibition pathway.
AuthorsMellet P, Bieth JG
JournalJ Biol Chem
PubMed ID10753871
'The inhibition of proteinases by serpins involves cleavage of the serpin, acylation, and translocation of the proteinase. To see whether acylation precedes or follows translocation, we have investigated the pH dependence of the interaction of fluorescein isothiocyanate-elastase with rhodamine alpha(1)-proteinase inhibitor (alpha(1)PI) using two independent methods: (i) kinetics of fluorescence ... More
Differential behavior of two cysteine residues on the myosin head in muscle fibers.
AuthorsMiyanishi T, Borejdo J
JournalBiochemistry
PubMed ID2523734
'We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SH1 thiol of S-1 moieties changes when MgADP is added to the fibers in rigor [Borejdo, J., Assulin, O., Ando, T., & Putnam, S. (1982) J. Mol. Biol. 158, 391-414. Burghardt, T.P., Ando, T., & Borejdo, J. (1983) Proc. ... More
Subunit interactions of GTP-binding proteins.
AuthorsHeithier H, Fröhlich M, Dees C, Baumann M, Häring M, Gierschik P, Schiltz E, Vaz WL, Hekman M, Helmreich EJ
JournalEur J Biochem
PubMed ID1312936
'Fluorescence energy transfer [cf. Förster, T. (1948) Ann. Phys. 6, 55-75] was tested for its suitability to study quantitative interactions of subunits of G0 with each other and these subunits or trimeric G0 with the beta 1-adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., ... More
Myosin II filament assemblies in the active lamella of fibroblasts: their morphogenesis and role in the formation of actin filament bundles.
AuthorsVerkhovsky AB, Svitkina TM, Borisy GG
JournalJ Cell Biol
PubMed ID7490299
'The morphogenesis of myosin II structures in active lamella undergoing net protrusion was analyzed by correlative fluorescence and electron microscopy. In rat embryo fibroblasts (REF 52) microinjected with tetramethylrhodamine-myosin II, nascent myosin spots formed close to the active edge during periods of retraction and then elongated into wavy ribbons of ... More
Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.
AuthorsLee NK, Kapanidis AN, Wang Y, Michalet X, Mukhopadhyay J, Ebright RH, Weiss S
JournalBiophys J
PubMed ID15653725
'Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies ... More
Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.
AuthorsBlanchoin L, Pollard TD
JournalJ Biol Chem
PubMed ID10336448
'We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle ... More
Sequence specific fluorescence detection of double strand DNA.
'Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide-fluorophore conjugates in ... More
Microscopic analysis of polymerization dynamics with individual actin filaments.
AuthorsFujiwara I, Takahashi S, Tadakuma H, Funatsu T, Ishiwata S
JournalNat Cell Biol
PubMed ID12198494
'The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and ... More
Analysis of biomolecular interactions using a miniaturized surface plasmon resonance sensor.
'A commercially available miniaturized surface plasmon resonance sensor has been investigated for its applicability to biological interaction analysis. The sensor was found to exhibit excellent repeatability and linearity for high-refractive index solutions and good reproducibility for the binding of proteins. Its detection limit for the monoclonal antibody M1 was found ... More
Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel.
AuthorsGlauner KS, Mannuzzu LM, Gandhi CS, Isacoff EY
JournalNature
PubMed ID10617202
'Voltage-gated ion channels underlie the generation of action potentials and trigger neurosecretion and muscle contraction. These channels consist of an inner pore-forming domain, which contains the ion permeation pathway and elements of its gates, together with four voltage-sensing domains, which regulate the gates. To understand the mechanism of voltage sensing ... More
An intermediate state of the gamma-aminobutyric acid transporter GAT1 revealed by simultaneous voltage clamp and fluorescence.
AuthorsLi M, Farley RA, Lester HA
JournalJ Gen Physiol
PubMed ID10736315
'The rat gamma-aminobutyric acid transporter GAT1 expressed in Xenopus oocytes was labeled at Cys74, and at one or more other sites, by tetramethylrhodamine-5-maleimide, without significantly altering GAT1 function. Voltage-jump relaxation analysis showed that fluorescence increased slightly and monotonically with hyperpolarization; the fluorescence at -140 mV was approximately 0. 8% greater ... More
Self-assembling light-harvesting systems from synthetically modified tobacco mosaic virus coat proteins.
AuthorsMiller RA, Presley AD, Francis MB
JournalJ Am Chem Soc
PubMed ID17319656
'A new protein-based approach has been developed for the construction of light-harvesting systems through self-assembly. The building blocks were prepared by attaching fluorescent chromophores to cysteine residues introduced on tobacco mosaic virus coat protein monomers. When placed under the appropriate buffer conditions, these conjugates could be assembled into stacks of ... More
Spectral fluctuation of a single fluorophore conjugated to a protein molecule.
AuthorsWazawa T, Ishii Y, Funatsu T, Yanagida T
JournalBiophys J
PubMed ID10692340
'We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous ... More
Structural rearrangements in single ion channels detected optically in living cells.
AuthorsSonnleitner A, Mannuzzu LM, Terakawa S, Isacoff EY
JournalProc Natl Acad Sci U S A
PubMed ID12228726
'Total internal reflection fluorescence microscopy was used to detect single fluorescently labeled voltage-gated Shaker K(+) channels in the plasma membrane of living cells. Tetramethylrhodamine (TMR) attached to specific amino acid positions in the voltage-sensing S4 segment changed fluorescence intensity in response to the voltage-driven protein motions of the channel. The ... More
Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation.
AuthorsMehan RS, White NC, Falke JJ
JournalBiochemistry
PubMed ID12627961
'The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and ... More
Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase.
'In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies ... More
Fast and slow voltage sensor movements in HERG potassium channels.
AuthorsSmith PL, Yellen G
JournalJ Gen Physiol
PubMed ID11865022
'HERG encodes an inwardly-rectifying potassium channel that plays an important role in repolarization of the cardiac action potential. Inward rectification of HERG channels results from rapid and voltage-dependent inactivation gating, combined with very slow activation gating. We asked whether the voltage sensor is implicated in the unusual properties of HERG ... More
Tight steric closure at the intracellular activation gate of a voltage-gated K(+) channel.
Authorsdel Camino D, Yellen G
JournalNeuron
PubMed ID11719205
'In voltage-gated K(+) channels (Kv), an intracellular gate regulates access from the cytoplasm to the pore by organic channel blockers and by chemical modifiers. But is ion flow itself controlled instead by constriction of the narrow selectivity filter near the extracellular surface? We find that the intracellular gate of Kv ... More
Intramolecular distances within the Ca(2+)-ATPase from sarcoplasmic reticulum as estimated through fluorescence energy transfer between probes.
'Fluorescence energy transfer measurements have been carried out to estimate intramolecular distances between probes bound to Ca(2+)-transporting ATPase (Ca(2+)-ATPase) as well as distances between these probes and the phospholipid headgroup. The nucleotide binding site was monitored by using 1,N6-ethenoadenosine 5''-triphosphate, a fluorescent analogue of ATP, and also by labelling Lys515 ... More
Novel tools for the study of development, migration and turnover of nematocytes (cnidarian stinging cells).
AuthorsWeber J
JournalJ Cell Sci
PubMed ID7738115
'The rhodamine derivatives tetramethyl-rhodamine-5/6-maleimide (TROMI) and tetramethyl-rhodamine-6-isothiocyanate (TRITC) were allowed to react with living Hydra vulgaris. The two fluorescent dyes stain the polyps to different degrees, apparently without impairing their viability and behaviour. Concerning nematocytes, TROMI preferentially couples to cytoskeletal elements only of mounted nematocytes whereas TRITC selectively reacts with ... More
Voltage clamp fluorimetry reveals a novel outer pore instability in a mammalian voltage-gated potassium channel.
AuthorsVaid M, Claydon TW, Rezazadeh S, Fedida D,
JournalJ Gen Physiol
PubMed ID18625849
'Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K(+) ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study ... More
Interaction of actin monomers with Acanthamoeba actophorin (ADF/cofilin) and profilin.
AuthorsBlanchoin L, Pollard TD
JournalJ Biol Chem
PubMed ID9737968
'Acanthamoeba actophorin is a member of ADF/cofilin family that binds both actin monomers and filaments. We used fluorescence anisotropy to study the interaction of actin monomers with recombinant actophorin labeled with rhodamine on a cysteine substituted for Serine-88. Labeled actophorin retains its affinity for actin and ability to reduce the ... More
Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix.
AuthorsReshetnyak YK, Andreev OA, Lehnert U, Engelman DM
JournalProc Natl Acad Sci U S A
PubMed ID16608910
'We have previously observed the spontaneous, pH-dependent insertion of a water-soluble peptide to form a helix across lipid bilayers [Hunt, J. F., Rath, P., Rothschild, K. J. & Engelman, D. M. (1997) Biochemistry 36, 15177-15192]. We now use a related peptide, pH (low) insertion peptide, to translocate cargo molecules attached ... More
Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms.
AuthorsSmith JJ, Conrad DW, Cuneo MJ, Hellinga HW
JournalProtein Sci
PubMed ID15576565
'Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We ... More
Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging.
AuthorsYang J, Chen H, Vlahov IR, Cheng JX, Low PS
JournalProc Natl Acad Sci U S A
PubMed ID16950881
'Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to ... More
Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA.
AuthorsMukhopadhyay J, Kapanidis AN, Mekler V, Kortkhonjia E, Ebright YW, Ebright RH
JournalCell
PubMed ID11525731
'Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition ... More
The crystal structure of uncomplexed actin in the ADP state.
AuthorsOtterbein LR, Graceffa P, Dominguez R
JournalScience
PubMed ID11474115
The dynamics and polarity of actin filaments are controlled by a conformational change coupled to the hydrolysis of adenosine 5'-triphosphate (ATP) by a mechanism that remains to be elucidated. Actin modified to block polymerization was crystallized in the adenosine 5'-diphosphate (ADP) state, and the structure was solved to 1.54 angstrom ... More
Direct physical measure of conformational rearrangement underlying potassium channel gating.
AuthorsMannuzzu LM, Moronne MM, Isacoff EY
JournalScience
PubMed ID8539623
In response to membrane depolarization, voltage-gated ion channels undergo a structural rearrangement that moves charges or dipoles in the membrane electric field and opens the channel-conducting pathway. By combination of site-specific fluorescent labeling of the Shaker potassium channel protein with voltage clamping, this gating conformational change was measured in real ... More
Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation.
AuthorsCha A, Ruben PC, George AL, Fujimoto E, Bezanilla F
JournalNeuron
PubMed ID10027291
Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 ... More
Photoaffinity labeling of antibodies for applications in homogeneous fluoroimmunoassays.
AuthorsChang IN, Lin JN, Andrade JD, Herron JN
JournalAnal Chem
PubMed ID7762830
A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and ... More
The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain.
AuthorsYou M, Li E, Hristova K
JournalBiochemistry
PubMed ID16634636
The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to ... More
Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism.
AuthorsMcGeoch AT, Trakselis MA, Laskey RA, Bell SD
JournalNat Struct Mol Biol
PubMed ID16116441
The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the ... More
The voltage-clamp fluorometry technique.
AuthorsGandhi CS, Olcese R,
JournalMethods Mol Biol
PubMed ID18998096
Ion channels are the cell's gatekeepers. These proteins selectively allow ionic current to flow down its electrochemical gradient. In some cases, specialized chemical or voltage sensing domains respond to environmental changes and signal the cell to adjust its internal chemistry in response to its surroundings. Because of their importance in ... More
Pathway of actin filament branch formation by Arp2/3 complex.
AuthorsBeltzner CC, Pollard TD,
JournalJ Biol Chem
PubMed ID18165685
A spectroscopic assay using pyrene-labeled fission yeast Arp2/3 complex revealed that the complex binds to and dissociates from actin filaments extremely slowly with or without the nucleation-promoting factor fission yeast Wsp1-VCA. Wsp1-VCA binds both Arp2/3 complex and actin monomers with high affinity. These two ligands have only modest impacts on ... More
Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein.
AuthorsSuzuki M, Ito Y, Sakata I, Sakai T, Husimi Y, Douglas KT,
JournalBiochem Biophys Res Commun
PubMed ID15796904
Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated ... More
Evaluation of three-dimensional microchannel glass biochips for multiplexed nucleic acid fluorescence hybridization assays.
AuthorsBenoit V, Steel A, Torres M, Yu YY, Yang H, Cooper J
JournalAnal Chem
PubMed ID11403280
Three-dimensional, flow-through microchannel glass substrates have a potential for enhanced performance, including increased sensitivity and dynamic range, over traditional planar substrates used in medium-density microarray platforms. This paper presents a methodology for the implementation of multiplexed nucleic acid hybridization fluorescence assays on microchannel glass substrates. Fluorescence detection was achieved, in ... More
Voltage-dependent conformational changes in human Ca(2+)- and voltage-activated K(+) channel, revealed by voltage-clamp fluorometry.
AuthorsSavalli N, Kondratiev A, Toro L, Olcese R
JournalProc Natl Acad Sci U S A
PubMed ID16895996
Large conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels regulate important physiological processes such as neurotransmitter release and vascular tone. BK(Ca) channels possess a voltage sensor mainly represented by the S4 transmembrane domain. Changes in membrane potential displace the voltage sensor, producing a conformational change that leads to channel opening. By ... More
Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds.
AuthorsMandato CA, Bement WM
JournalJ Cell Biol
PubMed ID11502762
Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic "contractile ring" composed of F-actin and myosin 2. However, this ring works in concert ... More
Measuring kinesin's first step.
AuthorsRosenfeld SS, Xing J, Jefferson GM, Cheung HC, King PH
JournalJ Biol Chem
PubMed ID12122000
A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along ... More
Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase.
AuthorsZimmermann B, Diez M, Zarrabi N, Gräber P, Börsch M
JournalEMBO J
PubMed ID15920483
F0F1-ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the epsilon-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence-labeled F0F1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the epsilon-subunit rotates stepwise ... More
Voltage-clamp fluorometry in the local environment of the C255-C511 disulfide bridge of the Na+/glucose cotransporter.
AuthorsGagnon DG, Frindel C, Lapointe JY
JournalBiophys J
PubMed ID17208964
We recently identified a functionally important disulfide bridge between C255 and C511 of the human Na+/glucose cotransporter SGLT1. In this study, voltage-clamp fluorometry was used to characterize the fluorescence of four different dyes attached to C255 and C511 under various ionic and substrate/inhibitor conditions. State-dependent fluorescence changes (DeltaF) were observed ... More
Effects of elevated intracellular magnesium on cytoskeletal integrity.
Increasing the intracellular magnesium concentration of PtK2 cells by 1 mM or more resulted in the disassembly of the interphase microtubule array over a period of 5 min after microinjection. This effect was found to be both transient and fully reversible, with the microtubule arrays reforming after further incubation. These ... More
Activation of the Arp2/3 complex by the Listeria acta protein. Acta binds two actin monomers and three subunits of the Arp2/3 complex.
AuthorsZalevsky J, Grigorova I, Mullins RD
JournalJ Biol Chem
PubMed ID11029465
ActA is a bacterially encoded protein that enables Listeria monocytogenes to hijack the host cell actin cytoskeleton. It promotes Arp2/3-dependent actin nucleation, but its interactions with cellular components of the nucleation machinery are not well understood. Here we show that two domains of ActA (residues 85-104 and 121-138) with sequence ... More
On the stator of rotary ATP synthase: the binding strength of subunit delta to (alpha beta)3 as determined by fluorescence correlation spectroscopy.
AuthorsHäsler K, Pänke O, Junge W
JournalBiochemistry
PubMed ID10521283
ATP synthase is conceived as a rotary enzyme. Proton flow drives the rotor (namely, subunits c12 epsilon gamma) relative to the stator (namely, subunits ab2 delta(alpha beta)3) and extrudes spontaneously formed ATP from three symmetrically arranged binding sites on (alpha beta)3 into the solution. We asked whether the binding of ... More
Stopped flow fluorescence energy transfer measurement of the rate constants describing the reversible formation and the irreversible rearrangement of the elastase-alpha1-proteinase inhibitor complex.
AuthorsMellet P, Boudier C, Mely Y, Bieth JG
JournalJ Biol Chem
PubMed ID9535901
Serpins are thought to inhibit proteinases by first forming a Michaelis-type complex that later converts into a stable inhibitory species. However, there is only circumstantial evidence for such a two-step reaction pathway. Here we directly observe the sequential appearance of two complexes by measuring the time-dependent change in fluorescence resonance ... More
Arp2/3 complex from Acanthamoeba binds profilin and cross-links actin filaments.
AuthorsMullins RD, Kelleher JF, Xu J, Pollard TD
JournalMol Biol Cell
PubMed ID9529382
The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site ... More
Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes.
AuthorsWard LD, Cantrill RC, Heithier H, Peters R, Helmreich EJ
JournalBiochim Biophys Acta
PubMed ID2844292
When monolayer cultured hepatocytes were incubated with 1 nM [125I]glucagon at 30 degrees C, equilibrium was reached after 10 min, whereas at 4 degrees C, equilibrium was reached after 60 min. At the higher temperature, 11.2% of the bound ligand was broken down after 60 min, at the lower temperature, ... More
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and forster resonance energy transfer suggest weak interactions between fibroblast growth factor receptor 3 (FGFR3) transmembrane domains in the absence of extracellular domains and ligands.
AuthorsLi E, You M, Hristova K
JournalBiochemistry
PubMed ID15628877
Lateral dimerization of membrane proteins has evolved as a means of signal transduction across the plasma membrane for all receptor tyrosine kinases (RTKs). The transmembrane (TM) domains of RTKs are proposed to play an important role in the dimerization process. We have investigated whether the TM domains of one RTK, ... More
The role of the betaDELSEED motif of F1-ATPase: propagation of the inhibitory effect of the epsilon subunit.
AuthorsHara KY, Kato-Yamada Y, Kikuchi Y, Hisabori T, Yoshida M
JournalJ Biol Chem
PubMed ID11279233
In F(1)-ATPase, a rotary motor enzyme, the region of the conserved DELSEED motif in the beta subunit moves and contacts the rotor gamma subunit when the nucleotide fills the catalytic site, and the acidic nature of the motif was previously assumed to play a critical role in rotation. Our previous ... More
Studies of ligand binding to Escherichia coli adenylosuccinate synthetase.
AuthorsSoans C, Fromm HJ
JournalArch Biochem Biophys
PubMed ID1929424
Dissociation constants of Escherichia coli adenylosuccinate synthetase with IMP, GTP, adenylosuccinate, and AMP (a competitive inhibitor for IMP) were determined by measuring the extent of quenching of the intrinsic tryptophan fluorescence of the enzyme. The enzyme has one binding site for each of these ligands. Aspartate and GDP did not ... More
Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes.
AuthorsMargeat E, Kapanidis AN, Tinnefeld P, Wang Y, Mukhopadhyay J, Ebright RH, Weiss S
JournalBiophys J
PubMed ID16299085
Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We ... More
Design of helical proteins for real-time endoprotease assays.
AuthorsSteinrücke P, Aldinger U, Hill O, Hillisch A, Basch R, Diekmann S
JournalAnal Biochem
PubMed ID11038269
Proteases play a key role in cellular biology and have become priority targets for new pharmaceuticals. Thus, there is a high demand for specific, sensitive, and quick assays to monitor the activity of endoproteases. We designed affinity-tagged helical proteins with unique protease cleavage sites and thus constructed universal, molecularly defined, ... More
Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states.
AuthorsSwaminath G, Xiang Y, Lee TW, Steenhuis J, Parnot C, Kobilka BK
JournalJ Biol Chem
PubMed ID14559905
The beta2 adrenoreceptor (beta2AR) is a prototypical G protein-coupled receptor (GPCR) activated by catecholamines. Agonist activation of GPCRs leads to sequential interactions with heterotrimeric G proteins, which activate cellular signaling cascades, and with GPCR kinases and arrestins, which attenuate GPCR-mediated signaling. We used fluorescence spectroscopy to monitor catecholamine-induced conformational changes ... More
Interactions of Acanthamoeba profilin with actin and nucleotides bound to actin.
AuthorsVinson VK, De La Cruz EM, Higgs HN, Pollard TD
JournalBiochemistry
PubMed ID9692980
Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of ... More
Profilin is predominantly associated with monomeric actin in Acanthamoeba.
AuthorsKaiser DA, Vinson VK, Murphy DB, Pollard TD
JournalJ Cell Sci
PubMed ID10523513
We used biochemical fractionation, immunoassays and microscopy of live and fixed Acanthamoeba to determine how much profilin is bound to its known ligands: actin, membrane PIP(2), Arp2/3 complex and polyproline sequences. Virtually all profilin is soluble after gentle homogenization of cells. During gel filtration of extracts on Sephadex G75, approximately ... More
Preparation of bead-tailed actin filaments: estimation of the torque produced by the sliding force in an in vitro motility assay.
AuthorsSuzuki N, Miyata H, Ishiwata S, Kinosita K
JournalBiophys J
PubMed ID8770216
By coating covalently the surface of a polystyrene bead (diameter = 1 micron) with gelsolin, we have succeeded in attaching the bead selectively at the barbed end of an actin filament and forming a 1:1 bead-actin filament complex. On a layer of heavy meromyosin on a nitrocellulose-coated coverglass, this bead-actin ... More
Actin-latrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A.
AuthorsYarmola EG, Somasundaram T, Boring TA, Spector I, Bubb MR
JournalJ Biol Chem
PubMed ID10859320
Latrunculin A is used extensively as an agent to sequester monomeric actin in living cells. We hypothesize that additional activities of latrunculin A may be important for its biological activity. Our data are consistent with the formation of a 1:1 stoichiometric complex with an equilibrium dissociation constant of 0.2 to ... More
Specific cross-linking of the SH1 thiol of skeletal myosin subfragment 1 to F-actin and G-actin.
AuthorsBettache N, Bertrand R, Kassab R
JournalBiochemistry
PubMed ID1731896
Recently, we reported that (maleimidobenzoyl)-G-actin (MBS-G-actin), which was resistant to the salt and myosin subfragment 1 (S-1) induced polymerizations, reacts reversibly and covalently in solution with the S-1 heavy chain at or near the strong F-actin binding region [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. ... More
Use of synthetic peptides as tracer antigens in fluorescence polarization immunoassays of high molecular weight analytes.
AuthorsWei AP, Herron JN
JournalAnal Chem
PubMed ID8297026
This paper describes a homogeneous immunoassay based on fluorescence polarization that enables subnanomolar detection of high molecular weight analytes. A monoclonal antibody (Mab) to human chorionic gonadotrophin (hCG) was screened against a panel of 221 synthetic peptides using the method of Geysen et al. (Geysen, H. M.; et al. J. ... More
Motor function and regulation of myosin X.
AuthorsHomma K, Saito J, Ikebe R, Ikebe M
JournalJ Biol Chem
PubMed ID11457842
Myosin X is a member of the diverse myosin superfamily that is ubiquitously expressed in various mammalian tissues. Although its association with actin in cells has been shown, little is known about its biochemical and mechanoenzymatic function at the molecular level. We expressed bovine myosin X containing the entire head, ... More
Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer.
AuthorsRichardson J, Blunck R, Ge P, Selvin PR, Bezanilla F, Papazian DM, Correa AM
JournalProc Natl Acad Sci U S A
PubMed ID17043236
Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal ... More
Movement of the helical domain of the epsilon subunit is required for the activation of thermophilic F1-ATPase.
AuthorsKato-Yamada Y, Yoshida M, Hisabori T
JournalJ Biol Chem
PubMed ID10958801
The inhibitory effect of epsilon subunit in F(1)-ATPase from thermophilic Bacillus PS3 was examined focusing on the structure-function relationship. For this purpose, we designed a mutant for epsilon subunit similar to the one constructed by Schulenberg and Capaldi (Schulenberg, B., and Capaldi, R. A. (1999) J. Biol. Chem. 274, 28351-28355). ... More
Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes.
AuthorsWenz JJ, Barrantes FJ
JournalBiochemistry
PubMed ID15628882
Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of ... More
Binding of the b-subunit in the ATP synthase from Escherichia coli.
AuthorsDiez M, Börsch M, Zimmermann B, Turina P, Dunn SD, Gräber P
JournalBiochemistry
PubMed ID14744151
The rotary mechanism of ATP synthase requires a strong binding within stator subunits. In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant ... More
CheA Kinase of bacterial chemotaxis: chemical mapping of four essential docking sites.
AuthorsMiller AS, Kohout SC, Gilman KA, Falke JJ
JournalBiochemistry
PubMed ID16846213
The chemotaxis pathway of Escherichia coli and Salmonella typhimurium is the paradigm for the ubiquitous class of 2-component signaling pathways in prokaryotic organisms. Chemosensing begins with the binding of a chemical attractant to a transmembrane receptor on the cell surface. The resulting transmembrane signal regulates a cytoplasmic, multiprotein signaling complex ... More
Structural implications of fluorescence quenching in the Shaker K+ channel.
AuthorsCha A, Bezanilla F
JournalJ Gen Physiol
PubMed ID9758859
When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213-216; Cha, A., and F. Bezanilla. ... More
Analysis of tetramethylrhodamine-labeled actin polymerization and interaction with actin regulatory proteins.
AuthorsPelikan Conchaudron A, Didry D, Le KH, Larquet E, Boisset N, Pantaloni D, Carlier MF
JournalJ Biol Chem
PubMed ID16757474
The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling ... More
Coexisting conformations of fibronectin in cell culture imaged using fluorescence resonance energy transfer.
AuthorsBaneyx G, Baugh L, Vogel V
JournalProc Natl Acad Sci U S A
PubMed ID11717404
Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET ... More
Dye-pair reporter systems for protein-peptide molecular interactions.
AuthorsGeoghegan KF, Rosner PJ, Hoth LR
JournalBioconjug Chem
PubMed ID10639088
Modifying a linear peptide near each terminus with a fluorescent dye can make it able to signal its own binding to a protein. As originally described, the dye pair is composed of fluorescein and tetramethylrhodamine [Wei, A.-P., Blumenthal, D. K., and Herron, J. N. (1994) Anal. Chem. 66, 1500-1506]. This ... More
Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion.
At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not ... More
Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives.
AuthorsHeithier H, Ward LD, Cantrill RC, Klein HW, Im MJ, Pollak G, Freeman B, Schiltz E, Peters R, Helmreich EJ
JournalBiochim Biophys Acta
PubMed ID2844291
The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), ... More
Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.
AuthorsWei AP, Blumenthal DK, Herron JN
JournalAnal Chem
PubMed ID7517105
A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and ... More
Fluorescence energy transfer between the primer and the beta subunit of the DNA polymerase III holoenzyme.
AuthorsGriep MA, McHenry CS
JournalJ Biol Chem
PubMed ID1737760
We report here our initial success in using fluorescence energy transfer to map the position of the subunits of the DNA polymerase III holoenzyme within initiation complexes formed on primed DNA. Using primers containing a fluorescent derivative 3 nucleotides from the 3'-terminus and acceptors of fluorescence energy transfer located on ... More
Conformational dynamics of the Na+/K+-ATPase probed by voltage clamp fluorometry.
AuthorsGeibel S, Kaplan JH, Bamberg E, Friedrich T
JournalProc Natl Acad Sci U S A
PubMed ID12552111
The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 ... More