Rapid binding of synapsin I to F- and G-actin. A study using fluorescence resonance energy transfer.
AuthorsCeccaldi PE, Benfenati F, Chieregatti E, Greengard P, Valtorta F
JournalFEBS Lett
PubMed ID8365471
Synapsin I is a nerve terminal phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation-dependent manner. By using fluorescence resonance energy transfer between purified components labeled with fluorescent probes, we now show that the binding of synapsin I to actin is a rapid phenomenon. Binding of synapsin I ... More
A non-radioactive automated method for DNA sequence determination.
AuthorsAnsorge W, Sproat BS, Stegemann J, Schwager C
JournalJ Biochem Biophys Methods
PubMed ID3559035
A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In ... More
Functional studies of the domains of talin.
AuthorsNuckolls GH, Turner CE, Burridge K
JournalJ Cell Biol
PubMed ID2110569
'The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments ... More
Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis.
AuthorsObinata T, Nagaoka-Yasuda R, Ono S, Kusano K, Mohri K, Ohtaka Y, Yamashiro S, Okada K, Abe H
JournalCell Struct Funct
PubMed ID9113405
'We previously demonstrated that small G-actin binding proteins, cofilin, ADF and profilin, are involved in the actin dynamics during myofibrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol., 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by ... More
Association of microinjected myosin and its subfragments with myofibrils in living muscle cells.
AuthorsJohnson CS, McKenna NM, Wang Y
JournalJ Cell Biol
PubMed ID3058721
'Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10-15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested ... More
Microinjection of nonmuscle and smooth muscle caldesmon into fibroblasts and muscle cells.
AuthorsYamakita Y, Yamashiro S, Matsumura F
JournalJ Cell Biol
PubMed ID2277070
'Caldesmon is present in a high molecular mass form in smooth muscle and predominantly in a low molecular mass form in nonmuscle cells. Their biochemical properties are very similar. To examine whether these two forms of caldesmon behave differently in cultured cells, we microinjected fluorescently labeled smooth muscle and nonmuscle ... 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
A kinetic mechanism for the polymerization of alpha1-antitrypsin.
AuthorsDafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas DA
JournalJ Biol Chem
PubMed ID10092640
'The mutation in the Z deficiency variant of alpha1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer ... More
Visualization of myosin in living cells.
AuthorsMittal B, Sanger JM, Sanger JW
JournalJ Cell Biol
PubMed ID3667695
'Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In ... More
Exchange of actin subunits at the leading edge of living fibroblasts: possible role of treadmilling.
AuthorsWang YL
JournalJ Cell Biol
PubMed ID4040521
'Previous observations indicated that the lamellipodium ("leading edge") of fibroblasts contains a dense meshwork, as well as numerous bundles (microspikes) of actin filaments. Most, if not all, of the filaments have a uniform polarity, with the "barbed" end associated with the membrane. I investigated whether and how actin subunits exchange ... More
Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin.
AuthorsGraceffa P
JournalJ Biol Chem
PubMed ID8530428
'The NH2- and COOH-terminal domains of muscle caldesmon are separated by a long alpha-helical stretch. Cys-580, in the COOH-terminal domain, can be rapidly and efficiently disulfide-cross-linked to Cys-374 of actin by incubation with actin modified with 5,5''-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310). Upon ... More
A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein.
AuthorsOkoh MP, Hunter JL, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID17144669
'A novel biosensor for inorganic phosphate (Pi) has been developed based on the phosphate binding protein of Escherichia coli. Two cysteine mutations were introduced and labeled with 6-iodoacetamidotetramethylrhodamine. When physically close to each other and correctly oriented, two rhodamine dyes interact to form a noncovalent dimer. In this state, they ... More
A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement.
AuthorsIsaacs BS, Husten EJ, Esmon CT, Johnson AE
JournalBiochemistry
PubMed ID3768326
'The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not ... More
Luminescence resonance energy transfer measurements in myosin.
AuthorsBurmeister Getz E, Cooke R, Selvin PR
JournalBiophys J
PubMed ID9591671
'Myosin is thought to generate force by a rotation between the relative orientations of two domains. Direct measurements of distances between the domains could potentially confirm and quantify these conformational changes, but efforts have been hampered by the large distances involved. Here we show that luminescence resonance energy transfer (LRET), ... More
Global configuration of single titin molecules observed through chain-associated rhodamine dimers.
AuthorsGrama L, Somogyi B, Kellermayer MS
JournalProc Natl Acad Sci U S A
PubMed ID11717390
'The global configuration of individual, surface-adsorbed molecules of the giant muscle protein titin, labeled with rhodamine conjugates, was followed with confocal microscopy. Fluorescence-emission intensity was reduced because of self-quenching caused by the close spacing between rhodamine dye molecules that formed dimers. In the presence of chemical denaturants, fluorescence intensity increased, ... More
Orientation and three-dimensional organization of actin filaments in dividing cultured cells.
AuthorsFishkind DJ, Wang YL
JournalJ Cell Biol
PubMed ID8227144
'The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to ... More
Cross-linker dynamics determine the mechanical properties of actin gels.
AuthorsWachsstock DH, Schwarz WH, Pollard TD
JournalBiophys J
PubMed ID8011912
'To evaluate the contributions of cross-linker dynamics and polymer deformation to the frequency-dependent stiffness of actin filament gels, we compared the rheological properties of actin gels with three types of cross-linkers: a weak one, Acanthamoeba alpha-actinin (dissociation rate constant 5.2 s-1, association rate constant 1.1 x 10(6) M-1 s-1); a ... More
In vivo dynamics of myosin II in Dictyostelium by fluorescent analogue cytochemistry.
AuthorsChu Q, Fukui Y
JournalCell Motil Cytoskeleton
PubMed ID8913645
'We used fluorescent analogue cytochemistry to study in vivo dynamics of myosin II in Dictyostelium discoideum. We labeled myosin with biotin or tetramethyl-rhodamine iodoacetamide (IATR). The labeled myosin shows normal activities as reversible filament assembly and Ca2+ and actin-activatable Mg(2+)-ATPase. We used the biotin-myosin as a probe examining the effects ... More
The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin.
'The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate ... More
Lateral diffusion of membrane lipids and proteins during the cell cycle of neuroblastoma cells.
Authorsde Laat SW, van der Saag PT, Elson EL, Schlessinger J
JournalProc Natl Acad Sci U S A
PubMed ID6929504
'The fluorescence photobleaching recovery method has been used to determine the lateral mobilities of membrane lipids and proteins during the cell cycle of synchronized C1300 mouse neuroblastoma cells (clone Neuro-2A). As probes for lipid mobility, 3,3''-dioctadecylindocarbocyanine iodide and a fluorescein-labeled analog of ganglioside GM1 were used. Membrane proteins were labeled ... More
Pathogenic alpha 1-antitrypsin polymers are formed by reactive loop-beta-sheet A linkage.
AuthorsSivasothy P, Dafforn TR, Gettins PG, Lomas DA
JournalJ Biol Chem
PubMed ID10924508
'alpha(1)-Antitrypsin is the most abundant circulating protease inhibitor and the archetype of the serine protease inhibitor or serpin superfamily. Members of this family may be inactivated by point mutations that favor transition to a polymeric conformation. This polymeric conformation underlies diseases as diverse as alpha(1)-antitrypsin deficiency-related cirrhosis, thrombosis, angio-edema, and ... More
Dephosphorylated synapsin I anchors synaptic vesicles to actin cytoskeleton: an analysis by videomicroscopy.
AuthorsCeccaldi PE, Grohovaz F, Benfenati F, Chieregatti E, Greengard P, Valtorta F
JournalJ Cell Biol
PubMed ID7876313
'Synapsin I is a synaptic vesicle-associated protein which inhibits neurotransmitter release, an effect which is abolished upon its phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Based on indirect evidence, it was suggested that this effect on neurotransmitter release may be achieved by the reversible anchoring of synaptic vesicles ... More
Evidence for cross-bridge order in contraction of glycerinated skeletal muscle.
AuthorsBurghardt TP, Ando T, Borejdo J
JournalProc Natl Acad Sci U S A
PubMed ID6584869
'The linear dichroism of iodoacetylrhodamine labels attached to the single reactive thiol groups of myosin heads was measured to determine the spatial orientation of myosin cross-bridges in single glycerinated skeletal muscle fibers. We have shown previously that in rigor the chromophoric labels are well ordered and assume an orientation nearly ... More
tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells.
AuthorsMhlanga MM, Vargas DY, Fung CW, Kramer FR, Tyagi S
JournalNucleic Acids Res
PubMed ID15809226
'When oligonucleotide probes are microinjected into cells to image the distribution of RNAs, they are rapidly sequestered into the nucleus. As a result, it is difficult to detect mRNAs in the cytoplasm of living cells. We were able to overcome this process by attaching tRNA transcripts to the probes. We ... More
Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.
AuthorsBlackman MJ, Corrie JE, Croney JC, Kelly G, Eccleston JF, Jameson DM
JournalBiochemistry
PubMed ID12356327
'Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine ... More
Viscoelastic response of fibroblasts to tension transmitted through adherens junctions.
AuthorsRagsdale GK, Phelps J, Luby-Phelps K
JournalBiophys J
PubMed ID9370474
'Cytoplasmic deformation was monitored by observing the displacements of 200-nm green fluorescent beads microinjected into the cytoplasm of Swiss 3T3 fibroblasts. We noted a novel protrusion of nonruffling cell margins that was accompanied by axial flow of beads and cytoplasmic vesicles as far as 50 microm behind the protruding plasma ... More
Orientational changes of crossbridges during single turnover of ATP.
AuthorsBorejdo J, Akopova I
JournalBiophys J
PubMed ID12668452
'Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the alpha-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added ... More
Modulation of intramolecular interactions in superhelical DNA by curved sequences: a Monte Carlo simulation study.
'A Monte Carlo model for the generation of superhelical DNA structures at thermodynamic equilibrium (Klenin et al., 1991; Vologodskii et al., 1992) was modified to account for the presence of local curvature. Equilibrium ensembles of a 2700-bp DNA chain at linking number difference delta Lk = -15 were generated, with ... More
Incorporation of fluorescently labeled actin and tropomyosin into muscle cells.
'The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation ... More
Actin polymerization. The effect of brevin on filament size and rate of polymerization.
AuthorsDoi Y, Frieden C
JournalJ Biol Chem
PubMed ID6480587
'Fluorescent probes covalently bound to actin or to the actin binding protein, brevin, have been utilized to provide information about actin filaments formed in the presence of brevin as well as about the effect of brevin on the rate of polymerization. At actin to brevin ratios of 10:1 to 100:1, ... More
Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.
'When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells'' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, ... More
Stereospecific reaction of muscle fiber proteins with the 5' or 6' isomer of (iodoacetamido)tetramethylrhodamine.
'The labeling of muscle fiber proteins with iodoacetamido)tetramethylrhodamine (IATR) was reinvestigated with the purified 5' or 6' isomers of IATR. Both isomers modify the myosin heavy chain within the 20-kDa fragment of myosin subfragment 1 (S1) but with different rates, and only the 5'-IATR alters K(+)-EDTA- and Ca(2+)-activated ATPases. Absorption ... More
Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.
AuthorsAlexandrov K, Simon I, Yurchenko V, Iakovenko A, Rostkova E, Scheidig AJ, Goody RS
JournalEur J Biochem
PubMed ID10491170
'Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein ... More
Wavelength-shifting molecular beacons.
AuthorsTyagi S, Marras SA, Kramer FR
JournalNat Biotechnol
PubMed ID11062440
'We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light ... More
A two-dimensional view of the folding energy landscape of cytochrome c.
AuthorsWerner JH, Joggerst R, Dyer RB, Goodwin PM
JournalProc Natl Acad Sci U S A
PubMed ID16844777
'Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture ... More
Two-step binding mechanism of fibrinogen to alpha IIb beta 3 integrin reconstituted into planar lipid bilayers.
AuthorsMüller B, Zerwes HG, Tangemann K, Peter J, Engel J
JournalJ Biol Chem
PubMed ID8454652
'The platelet integrin alpha IIb beta 3 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/1,2-dimyristoylphosphatidy lglycerol vesicles after removal of the detergent by adsorption ... More
Kinetics of histone endocytosis in Chinese hamster ovary cells. A flow cytofluorometric analysis.
AuthorsMurphy RF, Jorgensen ED, Cantor CR
JournalJ Biol Chem
PubMed ID7056738
'The endocytosis of histones by cultured cells was examined by flow cytofluorometry. Monolayer cultures of Chinese hamster ovary cells were incubated with fluorescein-labeled histone for various periods of time and then trypsinized to remove surface-bound protein. Internalization followed first order kinetics with a half-time of 45 min, and was linear ... More
Detection of actin assembly by fluorescence energy transfer.
AuthorsTaylor DL, Reidler J, Spudich JA, Stryer L
JournalJ Cell Biol
PubMed ID6894758
'Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole ... More
Path and extent of cross-bridge rotation during muscle contraction.
AuthorsAjtai K, Toft DJ, Burghardt TP
JournalBiochemistry
PubMed ID8180161
'The angular distribution of myosin cross-bridges in muscle fibers was investigated in four physiological states using a multiple probe analysis of varied extrinsic probes of the cross-bridge [Burghardt & Ajtai (1994) Biochemistry (preceding paper in this issue)]. The analysis combines data of complementary techniques from different probes giving the highest ... More
Binding and distribution of fluorescently labeled filamin in permeabilized and living cells.
AuthorsMittal B, Sanger JM, Sanger JW
JournalCell Motil Cytoskeleton
PubMed ID3690693
'This study reports the first development of a fluorescently labeled filamin. Smooth muscle filamin was labeled with fluorescent dyes in order to study its interaction with stress fibers and myofibrils, both in living cells and in permeabilized cells. The labeled filamin bound to the Z bands of isolated cross-striated myofibrils ... More
Tertiary structural changes in the cleft containing the ATP sensitive tryptophan and reactive thiol are consistent with pivoting of the myosin heavy chain at Gly699.
AuthorsBurghardt TP, Garamszegi SP, Park S, Ajtai K
JournalBiochemistry
PubMed ID9609697
'The conformation of myosin subfragment 1 (S1) in the vicinity of the ATP sensitive tryptophan (Trp510) and the highly reactive thiol (SH1), both residing in the "probe-binding" cleft at the junction of the catalytic and lever arm domains, was studied to ascertain its role in the mechanism of energy transduction ... More
Binding of heavy-chain and essential light-chain 1 of S1 to actin depends on the degree of saturation of F-actin filaments with S1.
AuthorsAndreev OA, Borejdo J
JournalBiochemistry
PubMed ID7578092
'The interaction of heavy-chain isoforms of myosin subfragment-1 with actin was examined by cross-linking with carbodiimide (EDC). The heavy chain of S1 could be cross-linked to a single actin molecule through sites on either 20 or 50 kDa proteolytic domains, resulting in complexes which migrated in an 8% polyacrylamide gel ... More
Fluorescent labeling of cell surfaces.
AuthorsEdidin M
JournalMethods Cell Biol
PubMed ID2464121
The rate of MgADP binding to and dissociation from acto-S1.
AuthorsBorejdo J, Ando T, Burghardt TP
JournalBiochim Biophys Acta
PubMed ID3872135
'The rate of binding and dissociation of MgADP from its ternary complex with actin and S1 was measured by following the extent to which fixed concentrations of MgADP slow down MgATP-induced dissociation of acto-S1. The solution of the equations describing this process shows that at any MgADP concentration the apparent ... More
Orientation changes of fluorescent probes at five sites on the myosin regulatory light chain during contraction of single skeletal muscle fibres.
AuthorsSabido-David C, Hopkins SC, Saraswat LD, Lowey S, Goldman YE, Irving M
JournalJ Mol Biol
PubMed ID9642045
'Changes in the orientation of the myosin regulatory light chain (RLC) in single muscle fibres were measured using polarised fluorescence from acetamidotetramethylrhodamine (ATR). Mutants of chicken skeletal RLC containing single cysteine residues at positions 2, 73, 94, 126 and 155 were labelled with either the 5 or 6-isomer of iodo-ATR, ... More
A fluorescent, reagentless biosensor for ADP based on tetramethylrhodamine-labeled ParM.
AuthorsKunzelmann S, Webb MR,
JournalACS Chem Biol
PubMed ID20158267
'Fluorescence assays for ADP detection are of considerable current interest, both in basic research and in drug discovery, as they provide a generic method for measuring the activity of ATPases and kinases. The development of a novel fluorescent biosensor is described that is based on a tetramethylrhodamine-labeled, bacterial actin homologue, ... More
Synthetic peptide GRGDS induces dissociation of alpha-actinin and vinculin from the sites of focal contacts.
AuthorsStickel SK, Wang YL
JournalJ Cell Biol
PubMed ID3138248
'The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were ... More
GroEL stimulates protein folding through forced unfolding.
AuthorsLin Z, Madan D, Rye HS,
JournalNat Struct Mol Biol
PubMed ID18311152
'Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of ... More
Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.
AuthorsLing N, Shrimpton C, Sleep J, Kendrick-Jones J, Irving M
JournalBiophys J
PubMed ID8785344
'The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged ... More
Function-based isolation of novel enzymes from a large library.
'Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (2) a positively charged moiety (3) the ... More
Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.
AuthorsShepard AA, Dumka D, Akopova I, Talent J, Borejdo J
JournalJ Muscle Res Cell Motil
PubMed ID15711885
'The rotation of myosin heads and actin were measured simultaneously with an indicator of the enzymatic activity of myosin. To minimize complications due to averaging of signals from many molecules, the signal was measured in a small population residing in a femtoliter volume of a muscle fiber. The onset of ... More
Measuring orientation of actin filaments within a cell: orientation of actin in intestinal microvilli.
AuthorsBorejdo J, Burlacu S
JournalBiophys J
PubMed ID8369437
'Orientational distribution of actin filaments within a cell is an important determinant of cellular shape and motility. To map this distribution we developed a method of measuring local orientation of actin filaments. In this method actin filaments within cells are labeled with fluorescent phalloidin and are viewed at high magnification ... More
Fluorescent modification and orientation of myosin sulfhydryl 2 in skeletal muscle fibers.
AuthorsAjtai K, Burghardt TP
JournalBiochemistry
PubMed ID2524213
'We describe a protocol for the selective covalent labeling of the sulfhydryl 2 (SH2) on the myosin cross-bridge in glycerinated muscle fibers using the sulfhydryl-selective label 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD). The protocol promotes the specificity of IANBD by using the ability to protect sulfhydryl 1 (SH1) from modification by binding the cross-bridge ... More
Affinity probe capillary electrophoresis: analysis of recombinant human growth hormone with a fluorescent labeled antibody fragment.
AuthorsShimura K, Karger BL
JournalAnal Chem
PubMed ID8116876
'A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess ... More
Real time imaging of single fluorophores on moving actin with an epifluorescence microscope.
AuthorsSase I, Miyata H, Corrie JE, Craik JS, Kinosita K
JournalBiophys J
PubMed ID8527645
'Relatively simple modifications of an ordinary epifluorescence microscope have greatly reduced its background luminescence, allowing continuous and real time imaging of single fluorophores in an aqueous medium. Main modifications were changing the excitation light path and setting an aperture stop so that stray light does not scatter inside the microscope. ... More
Subcellular distribution of rhodamine-actin microinjected into living fibroblastic cells.
AuthorsGlacy SD
JournalJ Cell Biol
PubMed ID6684662
'The time course and pattern of incorporation of rhodamine-labeled actin microinjected into cultured fibroblastic cells were examined by fluorescence microscopy. Following microinjection, the fluorescent probe was incorporated rapidly into ruffling membranes, and within 5 min faintly fluorescent stress fibers were observed. Levels of fluorescence in ruffling membranes then tended to ... More
Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils.
AuthorsAndreev OA, Andreeva AL, Borejdo J
JournalBiophys J
PubMed ID8241383
'Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In ... More
Polymerization and gelation of actin studied by fluorescence photobleaching recovery.
AuthorsTait JF, Frieden C
JournalBiochemistry
PubMed ID7115692
Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore.
AuthorsBorejdo J, Assulin O, Ando T, Putnam S
JournalJ Mol Biol
PubMed ID6982344
Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine.
AuthorsTait JF, Frieden C
JournalArch Biochem Biophys
PubMed ID7103505
Preparation of a fluorescent analog: acetamidofluoresceinyl-labeled Dictyostelium discoideum alpha-actinin.
AuthorsSimon JR, Taylor DL
JournalMethods Enzymol
PubMed ID3029546
Structure of actin observed by fluorescence resonance energy transfer spectroscopy.
AuthorsMiki M, O'Donoghue SI, Dos Remedios CG
JournalJ Muscle Res Cell Motil
PubMed ID1534564
On the elastic properties of tetramethylrhodamine F-actin.
AuthorsCintio O, Adami R, Choquet D, Grazi E
JournalBiophys Chem
PubMed ID11583836
(Iodoacetamido)tetramethylrhodamine disrupts F-actin. At the 1:1 fluorophore to actin (as monomer) ratio approximately 80% of the protein becomes non-sedimentable. The fluorescent, non-sedimentable actin copolymerizes with G-actin to yield fluorescent filaments. The tensile strength of these filaments changes with the ratio of the fluorescent non-sedimentable actin to the G-actin, being 1.6 ... More
Kinetic studies of Fos.Jun.DNA complex formation: DNA binding prior to dimerization.
AuthorsKohler JJ, Schepartz A
JournalBiochemistry
PubMed ID11141063
The bZIP proteins Fos and Jun bind DNA rapidly and with high affinity, forming a heteromeric complex that mediates activated transcription. Here we use stopped-flow fluorescence resonance energy transfer (FRET) to study the kinetic pathway by which Fos.Jun. DNA complexes assemble. Though dimerization of Fos and Jun occurs rapidly in ... 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
A comparison of order and orientation of crossbridges in rigor and relaxed muscle fibres using fluorescence polarization.
AuthorsWilson MG, Mendelson RA
JournalJ Muscle Res Cell Motil
PubMed ID6668358
Information has been obtained concerning the spatial disposition of the fluorescent reagent 5-(iodoacetamidoethylaminonaphthalene)-1-sulphonic acid bound covalently to muscle proteins in chemically skinned fibres of rabbit psoas muscle, using a novel time-gated fluorescence detection system to reject scattered incident light selectively. The results are consistent with a model of muscle crossbridge ... More
Visualizing the distribution and transport of mRNAs in living cells.
AuthorsBratu DP, Cha BJ, Mhlanga MM, Kramer FR, Tyagi S
JournalProc Natl Acad Sci U S A
PubMed ID14583593
We have visualized the movements of native mRNAs in living cells. Using nuclease-resistant molecular beacons, we imaged the transport and localization of oskar mRNA in Drosophila melanogaster oocytes. When the localization pattern was altered by genetic manipulation of the mRNA's 3' untranslated region, or by chemical perturbation of the intracellular ... More
Evaluation of the binding of Acanthamoeba profilin to pyrene-labeled actin by fluorescence enhancement.
AuthorsLee S, Li M, Pollard TD
JournalAnal Biochem
PubMed ID3364709
We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodoacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enhanced up to ... More
Chemical modification of actin. Acceleration of polymerization and reduction of network formation by reaction with N-ethylmaleimide, (iodoacetamido)tetramethylrhodamine, or 7-chloro-4-nitro-2,1,3-benzoxadiazole.
AuthorsTait JF, Frieden C
JournalBiochemistry
PubMed ID7150541
We examined the properties of rabbit skeletal muscle actin labeled at Cys-373 with N-ethylmaleimide or with (iodoacetamido)tetramethylrhodamine, and of N-ethylmaleimide-actin further modified with 7-chloro-4-nitro-2,1,3-benzoxadiazole (which primarily labels Lys-372). All three derivatives polymerize more rapidly than unlabeled actin. As measured by fluorescence photobleaching recovery and low-shear viscometry, all three also show ... More
Isoenzyme-specific interaction of muscle-type creatine kinase with the sarcomeric M-line is mediated by NH(2)-terminal lysine charge-clamps.
AuthorsHornemann T, Stolz M, Wallimann T
JournalJ Cell Biol
PubMed ID10851020
Creatine kinase (CK) is located in an isoenzyme-specific manner at subcellular sites of energy production and consumption. In muscle cells, the muscle-type CK isoform (MM-CK) specifically interacts with the sarcomeric M-line, while the highly homologous brain-type CK isoform (BB-CK) does not share this property. Sequence comparison revealed two pairs of ... More
Dual-colour microscopy of single fluorophores bound to myosin interacting with fluorescently labelled actin using anti-Stokes fluorescence.
AuthorsSaito K, Tokunaga M, Iwane AH, Yanagida T
JournalJ Microsc
PubMed ID9450329
We have refined prismless total internal reflection fluorescence microscopy with extremely low background to visualize single fluorophores attached to protein molecules interacting with a filamentous biopolymer labelled with different colour fluorophores. By using Stokes and anti-Stokes fluorescence, two different colour fluorescences from two different colour fluorophores excited with a single ... More
Fluorescence polarization from isomers of tetramethylrhodamine at SH-1 in rabbit psoas muscle fibers.
AuthorsBerger CL, Craik JS, Trentham DR, Corrie JE, Goldman YE
JournalBiophys J
PubMed ID7787111
We have used fluorescence polarization to examine orientational changes of the 5- and 6-isomers of acetamidotetramethylrhodamine (ATR) covalently bound to SH-1 (Cys-707 of the myosin heavy chain) in single, skinned fibers from rabbit psoas muscle after rapid length steps or photolysis of caged nucleotides. Similar results were obtained with both ... More
The specific binding of small molecule isoprenoids to rhoGDP dissociation inhibitor (rhoGDI).
AuthorsMondal MS, Wang Z, Seeds AM, Rando RR
JournalBiochemistry
PubMed ID10631002
The activities of small G-proteins are in part regulated by their interactions with GDI proteins. This binding is thought to be dependent on the C-terminal isoprenoid modification (geranylgeranyl or farnesyl) of these proteins. G-proteins are generally isoprenylated/methylated at their C-terminal cysteine residues. A quantitative fluorescence assay is reported here to ... More
Cooperativity in F-actin: chemical modifications of actin monomers affect the functional interactions of myosin with unmodified monomers in the same actin filament.
AuthorsProchniewicz E, Katayama E, Yanagida T, Thomas DD
JournalBiophys J
PubMed ID8369420
We have chemically modified a fraction of the monomers in actin filaments, and then measured the effects on the functional interaction of myosin with unmodified monomers within the same filament. Two modifications were used: (a) covalent attachment of various amounts of myosin subfragment-1 (S1) with the bifunctional reagent disuccinimidyl suberate ... More
Formation and movement of myosin-containing structures in living fibroblasts.
AuthorsMcKenna NM, Wang YL, Konkel ME
JournalJ Cell Biol
PubMed ID2670956
Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, ... More
Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells.
AuthorsKolega J
JournalJ Cell Biochem
PubMed ID9518264
Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin ... More
Specific protein-membrane contacts are required for prepore and pore assembly by a cholesterol-dependent cytolysin.
AuthorsSoltani CE, Hotze EM, Johnson AE, Tweten RK
JournalJ Biol Chem
PubMed ID17412689
Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. ... More
Measurement of interprotein distances in the acto-subfragment 1 rigor complex.
AuthorsTakashi R, Kasprzak AA
JournalBiochemistry
PubMed ID3427088
Using enzymatic labeling, we have conjugated the fluorescence probe dansylcadaverine (DNC) to Gln-41 of rabbit skeletal muscle actin with the intention of utilizing the dansyl chromophore as a donor in fluorescence resonance energy transfer (FRET) distance measurements. The fluorescence decay of DNC-actin was found to consist of two decay constants ... More
Corequirement of specific phosphoinositides and small GTP-binding protein Cdc42 in inducing actin assembly in Xenopus egg extracts.
AuthorsMa L, Cantley LC, Janmey PA, Kirschner MW
JournalJ Cell Biol
PubMed ID9490725
Both phosphoinositides and small GTP-binding proteins of the Rho family have been postulated to regulate actin assembly in cells. We have reconstituted actin assembly in response to these signals in Xenopus extracts and examined the relationship of these pathways. We have found that GTPgammaS stimulates actin assembly in the presence ... More
Synthesis and applications of heterobifunctional photocleavable cross-linking reagents.
AuthorsMarriott G, Ottl J
JournalMethods Enzymol
PubMed ID9661150
This study designed, synthesized, and characterized a number of new heterobifunctional photocleavable cross-linking reagents that may be used to photomodulate the activity of proteins or to prepare caged fluorescent dyes. Biomolecules or fluorophores caged via a thiol group with the BNBASE reagent can be covalently linked to a second protein, ... More
Hepatitis B virus protein pX enhances the monomer assembly pathway of bZIP.DNA complexes.
AuthorsSchneider TL, Schepartz A
JournalBiochemistry
PubMed ID11258894
The rapid and correct assembly of dimeric transcription factors on target DNA is essential for accurate transcriptional regulation. Here we ask how a viral accessory factor, hepatitis B virus X protein (pX), influences the rate and identity of the assembly pathway followed by members of the basic region leucine zipper ... More
Fluorescence-labeled peptide pI markers for capillary isoelectric focusing.
AuthorsShimura K, Kamiya K, Matsumoto H, Kasai K
JournalAnal Chem
PubMed ID11924962
Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection. Tetra- to tridecapeptides containing one cysteine residue were designed to focus sharply at their respective isoelectric points by including amino acids that contain charged side chains, the pKa ... More
Diffusion of macromolecules and virus-like particles in human cervical mucus.
To determine whether or not large macromolecules and viruses can diffuse through mucus, we observed the motion of proteins, microspheres, and viruses in fresh samples of human cervical mucus using fluorescent recovery after photobleaching and multiple image photography. Two capsid virus-like particles, human papilloma virus (55 nm, approximately 20,000 kDa) ... More
Reorganization of alpha-actinin and vinculin induced by a phorbol ester in living cells.
AuthorsMeigs JB, Wang YL
JournalJ Cell Biol
PubMed ID3082892
We have used fluorescent analogue cytochemistry, image intensification, and digital image processing to examine the redistribution of alpha-actinin and vinculin in living cultured African green monkey kidney (BSC-1) cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Before treatment, microinjected alpha-actinin shows characteristic distribution along stress fibers and at adhesion plaques; ... More
Structure of the 265-kilodalton complex formed upon EDC cross-linking of subfragment 1 to F-actin.
AuthorsAndreeva AL, Andreev OA, Borejdo J
JournalBiochemistry
PubMed ID8268172
The conventional model of force generation in muscle requires the presence of at least two different contact areas between the myosin head (S1) and the actin filament. It has been found that S1 has two sites available for carbodiimide cross-linking, but it is generally believed that the myosin head can ... More
Diffusion of nerve growth factor in rat striatum as determined by multiphoton microscopy.
AuthorsStroh M, Zipfel WR, Williams RM, Webb WW, Saltzman WM
JournalBiophys J
PubMed ID12829512
Neurotrophins such as nerve growth factor (NGF) may be useful for treating diseases in the central nervous system; our ability to harness the potential therapeutic benefit of NGF is directly related to our understanding of the fate of exogenously supplied factors in brain tissue. We utilized multiphoton microscopy to quantify ... More
Preparation and incorporation of probe-labeled apoA-I for fluorescence resonance energy transfer studies of rHDL.
AuthorsLi HH, Thomas MJ, Pan W, Alexander E, Samuel M, Sorci-Thomas MG
JournalJ Lipid Res
PubMed ID11734582
Apolipoprotein A-I (apoA-I), the major constituent of HDL, plays an essential role in regulating cholesterol metabolism, acting as the physiological activator of lecithin: cholesterol acyltransferase, which converts cholesterol to cholesterol ester. Thiol-reactive fluorescent probes attached to cysteine-containing apoA-I mutants are currently being used to investigate the "LCAT active" conformation of ... More
Effect of ADP on binding of skeletal S1 to F-actin.
AuthorsAndreev OA, Ushakov DS, Borejdo J
JournalBiochemistry
PubMed ID9922150
The proximity of skeletal myosin subfragment-1 (S1) to actin, and its orientation with respect to thin filaments of single muscle fibers, were compared in the presence and in the absence of ADP. The proximity was assessed by the efficiency of carbodiimide-induced cross-linking and the orientation by polarization of fluorescence of ... More
Myosin essential light chain isoforms modulate the velocity of shortening propelled by nonphosphorylated cross-bridges.
AuthorsMatthew JD, Khromov AS, Trybus KM, Somlyo AP, Somlyo AV
JournalJ Biol Chem
PubMed ID9813037
The differential effects of essential light chain isoforms (LC17a and LC17b) on the mechanical properties of smooth muscle were determined by exchanging recombinant for endogenous LC17 in permeabilized smooth muscle treated with trifluoperazine (TFP). Co-precipitation with endogenous myosin heavy chain verified that 40-60% of endogenous LC17a could be exchanged for ... More
Conformational selection during weak binding at the actin and myosin interface.
AuthorsXu J, Root DD
JournalBiophys J
PubMed ID10969011
The molecular mechanism of the powerstroke in muscle is examined by resonance energy transfer techniques. Recent models suggesting a pre-cocking of the myosin head involving an enormous rotation between the lever arm and the catalytic domain were tested by measuring separation distances among myosin subfragment-2, the nucleotide site, and the ... More
Fluorescence resonance energy transfer measurements of distances in actin and myosin. A critical evaluation.
Authorsdos Remedios CG, Miki M, Barden JA
JournalJ Muscle Res Cell Motil
PubMed ID3298315
The contractile proteins actin and myosin are of considerable biological interest. They are essential for muscle contraction and in eukaryotic cells they play a crucial role in most contractile phenomena. Over the years since the first fluorescence resonance energy transfer (FRET) paper appeared, an extensive body of literature has accumulated ... More
Mechanical fatigue in repetitively stretched single molecules of titin.
AuthorsKellermayer MS, Smith SB, Bustamante C, Granzier HL
JournalBiophys J
PubMed ID11159452
Relaxed striated muscle cells exhibit mechanical fatigue when exposed to repeated stretch and release cycles. To understand the molecular basis of such mechanical fatigue, single molecules of the giant filamentous protein titin, which is the main determinant of sarcomeric elasticity, were repetitively stretched and released while their force response was ... More
Site-directed mutagenesis enabled preparation of a functional fluorescent analog of profilin: biochemical characterization and localization in living cells.
AuthorsTarachandani A, Wang YL
JournalCell Motil Cytoskeleton
PubMed ID8871818
The preparation of fluorescent profilin analogs for binding and spectroscopic studies, in vitro and in vivo, has been hampered by the poor chemical reactivity of this protein in its native form. We have addressed this problem by labeling a mutant, chemically reactive form of profilin. Site-directed mutagenesis was first used ... More
Mobility, clustering, and transport of nerve growth factor in embryonal sensory cells and in a sympathetic neuronal cell line.
AuthorsLevi A, Shechter Y, Neufeld EJ, Schlessinger J
JournalProc Natl Acad Sci U S A
PubMed ID6932032
We have prepared a fluorescent conjugate of nerve growth factor (NGF) containing 8--10 rhodamine molecules attached to free carboxyl groups of the protein. This analogue retained full binding capacity toward NGF receptors, full antigenic properties, and the potency to stimulate the differentiation of embryonal chicken sensory ganglia cells in vitro. ... More
A fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways.
AuthorsSato M, Ozawa T, Yoshida T, Umezawa Y
JournalAnal Chem
PubMed ID10500481
A fluorescent indicator for tyrosine phosphorylation-based insulin signaling is described. Upon binding of insulin to cell-surface insulin receptor, the receptor phosphorylates tyrosine residues of insulin receptor substrate 1 (IRS-1) in the cell. A fluorescent indicator was designed by using synthetic phosphopeptide pY939 derived from the tyrosine phosphorylation domain of IRS-1 ... More
Crossbridge order and orientation in resting single glycerinated muscle fibres studied by linear dichroism of bound rhodamine labels.
AuthorsBurghardt TP, Tidswell M, Borejdo J
JournalJ Muscle Res Cell Motil
PubMed ID6533157
Linear dichroism of iodoacetyl-rhodamine labels attached to the highly reactive thiol of the myosin heads was measured in order to infer the spatial orientation and the degree of order in myosin crossbridges in single glycerinated rabbit psoas fibres at rest. We have previously shown that in rigor the chromophoric labels ... More
Site-directed mutagenesis of the phosphorylation site of cofilin: its role in cofilin-actin interaction and cytoplasmic localization.
AuthorsNagaoka R, Abe H, Obinata T
JournalCell Motil Cytoskeleton
PubMed ID8913641
It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells ... 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
Possible translocation of actin and alpha-actinin along stress fibers.
AuthorsMcKenna NM, Wang YL
JournalExp Cell Res
PubMed ID3758212
We have employed fluorescent analogue cytochemistry and fluorescence photobleaching to study the mobility of actin and alpha-actin along stress fibers. Rhodamine-labeled actin or alpha-actinin microinjected into embryonic chick cardiac fibroblasts soon became incorporated into stress fibers. A pulse of a laser microbeam was used to photobleach small spots on the ... More
Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity.
AuthorsVaradarajan N, Gam J, Olsen MJ, Georgiou G, Iverson BL
JournalProc Natl Acad Sci U S A
PubMed ID15867160
The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower ... More