Dynamics of acrylodan-labeled bovine and human serum albumin entrapped in a sol-gel-derived biogel.
AuthorsJordan JD,Dunbar RA,Bright FV
JournalAnalytical chemistry
PubMed ID8686877
Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE.
AuthorsPierpaoli EV, Sandmeier E, Schönfeld HJ, Christen P
JournalJ Biol Chem
PubMed ID9506960
The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously ... More
Reagentless optical sensing of glutamine using a dual-emitting glutamine-binding protein.
AuthorsTolosa L, Ge X, Rao G
JournalAnal Biochem
PubMed ID12654305
Glutamine is a major source of nitrogen and carbon in cell culture media. Thus, glutamine monitoring is important in bioprocess control. Here we report a reagentless fluorescence sensing for glutamine based on the Escherichia coli glutamine-binding protein (GlnBP) that is sensitive in the submicromolar ranges. The S179C variant of GlnBP ... More
Fatty acid transport in adipocytes monitored by imaging intracellular free fatty acid levels.
AuthorsKampf JP, Kleinfeld AM
JournalJ Biol Chem
PubMed ID15199061
Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membrane's role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFA(i)]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFA(i)] was ... More
Actin can act as a cofactor for a viral proteinase in the cleavage of the cytoskeleton.
'Cytoskeletal proteins are exploited by many viruses during infection. We report a novel finding that actin can act as a cofactor for the adenovirus proteinase (AVP) in the degradation of cytoskeletal proteins. Transfection studies in HeLa cells revealed AVP localized with cytokeratin 18, and this was followed by destruction of ... More
Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin.
AuthorsGraceffa P
JournalBiochemistry
PubMed ID9092808
'Smooth muscle caldesmon is a single polypeptide chain with its NH2- and COOH-terminal domains separated by a long alpha-helix. Caldesmon was labeled at either Cys-153 in the NH2 domain or Cys-580 in the COOH domain with a variety of fluorescence probes. Fluorescence intensity, peak position, and polarization of probes on ... More
The pleckstrin homology domain of phospholipase Cbeta transmits enzymatic activation through modulation of the membrane-domain orientation.
AuthorsDrin G, Douguet D, Scarlata S
JournalBiochemistry
PubMed ID16669615
'Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also ... More
Membrane domains containing phosphatidylserine and substrate can be important for the activation of protein kinase C.
AuthorsYang L, Glaser M
JournalBiochemistry
PubMed ID7531492
'The relationship between lipid domains and enzyme activity was studied via the direct visualization and quantitation of domains by fluorescence digital imaging microscopy. The substrate used in these experiments was a basic peptide derived from a prominent cellular substrate (MARCKS) of protein kinase C. The MARCKS peptide and phosphatidylserine, which ... More
Close proximity of Cys64 and Cys140 in the delta subunit of Escherichia coli F1-ATPase.
AuthorsZiegler M, Xiao R, Penefsky HS
JournalJ Biol Chem
PubMed ID8307987
'The delta subunit of the F1-ATPase from Escherichia coli contains 2 cysteine residues, one at position 64 and the second at position 140 of the amino acid sequence. These residues were specifically labeled with sulfhydryl reagents in this study without labeling other -SH groups in the enzyme. Modification of Cys140 ... More
Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.
AuthorsYem AW, Epps DE, Mathews WR, Guido DM, Richard KA, Staite ND, Deibel MR
JournalJ Biol Chem
PubMed ID1531337
'Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate ... More
Probing the active center gorge of acetylcholinesterase by fluorophores linked to substituted cysteines.
AuthorsBoyd AE, Marnett AB, Wong L, Taylor P
JournalJ Biol Chem
PubMed ID10779503
'To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific ... More
Transformation of actin-encapsulating liposomes induced by cytochalasin D.
AuthorsMiyata H, Kinosita K
JournalBiophys J
PubMed ID7948706
'Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed ... More
Electron paramagnetic resonance of spin-labeled aequorin.
AuthorsKemple MD, Ray BD, Jarori GK, Rao BD, Prendergast FG
JournalBiochemistry
PubMed ID6487606
'Aequorin is a Ca-activated bioluminescent protein from jellyfish. This protein contains two sulfhydryl groups, one of which is essential for its bioluminescence. Little information concerning the structure of and relationship between the metal binding sites of aequorin and the sulfhydryl group(s) is known. Aequorin was modified by attachment of either ... More
Streptolysin O: inhibition of the conformational change during membrane binding of the monomer prevents oligomerization and pore formation.
AuthorsAbdel Ghani EM, Weis S, Walev I, Kehoe M, Bhakdi S, Palmer M
JournalBiochemistry
PubMed ID10563803
'Streptolysin O is a four-domain protein toxin that permeabilizes animal cell membranes. The toxin first binds as a monomer to membrane cholesterol and subsequently assembles into oligomeric transmembrane pores. Binding is mediated by a C-terminally located tryptophan-rich motif. In a previous study, conformational effects of membrane binding were characterized by ... More
Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9799521
'The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a ... More
Imaging of cAMP-dependent protein kinase activity in living neural cells using a novel fluorescent substrate.
AuthorsHigashi H, Sato K, Ohtake A, Omori A, Yoshida S, Kudo Y
JournalFEBS Lett
PubMed ID9305731
'In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable ... More
Use of the parallax-quench method to determine the position of the active-site loop of cholesterol oxidase in lipid bilayers.
AuthorsChen X, Wolfgang DE, Sampson NS
JournalBiochemistry
PubMed ID11063575
'To elucidate the cholesterol oxidase-membrane bilayer interaction, a cysteine was introduced into the active site lid at position-81 using the Brevibacterium enzyme. To eliminate the possibility of labeling native cysteine, the single cysteine in the wild-type enzyme was mutated to a serine without any change in activity. The loop-cysteine mutant ... More
In situ visualization of caspase-1-like activity associated with promotion of hippocampal cell death.
AuthorsNishii W, Shoda T, Matsumoto N, Nakamura T, Kudo Y, Takahashi K
JournalFEBS Lett
PubMed ID11997036
'To clarify the function of caspase-1-like proteases in neuronal cell death, it is important to be able to detect the activity in living organs by microscopic visualization. In the present study, we synthesized a novel fluorescent substrate sensitive to the caspase-1-like activity, which is easily introduced into cells constituting living ... More
Further study on the magnesium-mediated change in physical state of phospholipid modulates mitochondrial F0-F1-ATPase activity.
AuthorsHuang Y, Tong J, Liu Z, Yang F
JournalMagnes Res
PubMed ID8155482
'We have postulated that magnesium may play a role in altering the lipid fluidity of the bilayers, which would induce a change of conformation of the F0-ATPase portion (buried in the lipid core) of mitochondrial F0-F1-ATPase. Such change could be transmitted to the soluble F1 portion, resulting in higher enzymatic ... More
Very long chain n-3 and n-6 polyunsaturated fatty acids bind strongly to liver fatty acid-binding protein.
AuthorsNorris AW, Spector AA
JournalJ Lipid Res
PubMed ID11907148
'Synthesis of n-3 and n-6 very long chain-PUFAs (VLC-PUFAs) from 18-carbon essential fatty acids is differentially regulated. The predominant product arising from n-3 fatty acids is docosahexaenoic acid (22:6n-3), with the liver serving as the main site of production. The synthetic pathway requires movement of a 24-carbon intermediate from the ... More
Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate.
AuthorsShrestha S, Salins LL, Mark Ensor C, Daunert S
JournalBiotechnol Bioeng
PubMed ID12115121
'Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the ... More
Importance of the D and E helices of the molecular chaperone DnaK for ATP binding and substrate release.
AuthorsSlepenkov SV, Patchen B, Peterson KM, Witt SN
JournalBiochemistry
PubMed ID12741845
'The C-terminal domain of the molecular chaperone DnaK is a compact lid-like structure made up of five alpha-helices (alphaA-alphaE) (residues 508-608) that is followed by a 30-residue disordered, flexible region (609-638). The lid encapsulates the peptide molecule bound in the substrate-binding domain, whereas the function of the 30-residue disordered region ... More
Myosin S1 changes the orientation of caldesmon on actin.
AuthorsSzczesna D, Graceffa P, Wang CL, Lehrer SS
JournalBiochemistry
PubMed ID8204606
'Changes in the orientation of caldesmon bound to actin in skeletal ghost myofibrils caused by the binding of myosin subfragment 1 (S1) were measured by fluorescence-detected linear dichroism using fluorescence microscopy. Gizzard caldesmon, labeled with acrylodan at its two Cys residues (CaD*), bound to actin with a probe angle that ... More
Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases.
AuthorsOskolkova OV, Hermetter A
JournalBiochim Biophys Acta
PubMed ID12009403
'Triacylglycerol analogue p-nitrophenyl phosphonates specifically react with the active-site serine of lipolytic enzymes to give covalent lipase-inhibitor complexes, mimicking the first transition state which is involved in lipase-mediated ester hydrolysis. Here we report on a new type of phosphonate inhibitors containing a polarity-sensitive fluorophore to monitor micropolarity around the active ... More
Kinetics of binding of caldesmon to actin.
AuthorsChalovich JM, Chen YD, Dudek R, Luo H
JournalJ Biol Chem
PubMed ID7730374
'The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using ... More
Calcium-induced conformational change in cardiac troponin C studied by fluorescence probes attached to Cys-84.
AuthorsDong WJ, Cheung HC
JournalBiochim Biophys Acta
PubMed ID8695639
'Residue Cys-84 of bovine cardiac troponin C (cTnC) located at the C-terminal end of helix D was selectively labeled in the presence of Ca2+ with two fluorescent probes: IAANS (2-(4-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) and acrylodan (6-acrylol-2-(dimethylamino)naphthalene). The fluorescence of the attached probes was studied by the steady-state and time-resolved methods to gain ... More
Trehalose influence on beta-lactoglobulin stability and hydration by time resolved fluorescence.
AuthorsD'Alfonso L, Collini M, Baldini G
JournalEur J Biochem
PubMed ID12755705
'The stabilizing role of the disaccharide trehalose on beta-lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time-resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride ... More
Tyrosine phenol-lyase and tryptophan indole-lyase encapsulated in wet nanoporous silica gels: Selective stabilization of tertiary conformations.
'The pyridoxal 5''-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining ... More
Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe.
AuthorsTricerri MA, Behling Agree AK, Sanchez SA, Jonas A
JournalBiochemistry
PubMed ID11087425
'Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound ... More
'Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share ... More
Dimer-dimer interfaces of the lambda-repressor are different in liganded and free states.
AuthorsBandyopadhyay S, Mukhopadhyay C, Roy S
JournalBiochemistry
PubMed ID8664296
'Lambda-repressor dimers associate in solution to form tetramers and higher order structures. Dimer-dimer contact is also crucial in cooperative binding to adjacent operators. Fluorescence quenching studies indicate that the tryptophan 230 environment is significantly different in unliganded and adjacent operator-bound tetramers. Acrylodan attached to Cys 235, in a mutant F235C ... More
Fluorescence properties of acrylodan-labeled tropomyosin and tropomyosin-actin: evidence for myosin subfragment 1 induced changes in geometry between tropomyosin and actin.
AuthorsLehrer SS, Ishii Y
JournalBiochemistry
PubMed ID3191099
'The Cys groups of rabbit skeletal tropomyosin (Tm) and rabbit skeletal alpha alpha Tm were specifically labeled with acrylodan (AC). The probe on Tm is quite immobile yet exposed to solvent as indicated by its limiting polarization (P0 = 0.38) and fluorescence emission spectrum (lambda max = 520 nm) and ... More
Membrane binding of phospholipases C-beta 1 and C-beta 2 is independent of phosphatidylinositol 4,5-bisphosphate and the alpha and beta gamma subunits of G proteins.
AuthorsRunnels LW, Jenco J, Morris A, Scarlata S
JournalBiochemistry
PubMed ID8988021
'We have measured the membrane binding affinities of purified phosphatidylinositol-specific phospholipases C-beta 1 and C-beta 2 to membranes of varying lipid composition using fluorescence methods. Our studies show that these proteins bind with affinities of 10(-5)-10(-4) M, with a small dependence on lipid type. Binding was relatively insensitive to the ... More
Formation of membrane domains during the activation of protein kinase C.
AuthorsYang L, Glaser M
JournalBiochemistry
PubMed ID8909294
'The lateral membrane organization of phosphatidylserine, diacylglycerol, substrate, and Ca(2+)-dependent protein kinase C in large unilamellar vesicles was investigated by using fluorescence digital imaging microscopy. The formation of phosphatidylserine domains could be induced by either Ca2+, the MARCKS peptide, or protein kinase C. However, only Ca2+ could induce diacylglycerol to ... More
Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin.
'Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive ... More
Thermodynamics of substrate binding to the chaperone SecB.
AuthorsPanse VG, Swaminathan CP, Surolia A, Varadarajan R
JournalBiochemistry
PubMed ID10694412
'The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and alpha-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded ... More
Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors.
AuthorsRunnels LW, Scarlata SF
JournalBiochemistry
PubMed ID9931014
'Phosphatidylinositide-specific phospholipase C-betas play a key role in Ca2+ signaling and are specifically activated by the alphaq family of heterotrimeric G proteins and as well as betagamma subunits. We have determined the affinity between Gbetagamma subunits and GTPgammaS and GDP-liganded Galphaq subunits on membrane surfaces, and their respective affinities to ... More
Identification of the N-terminal peptide binding site of glucose-regulated protein 94.
'Because the stress protein GRP94 can augment presentation of peptides to T cells, it is important to define how it, as well as all other HSP90 family members, binds peptides. Having previously shown that the N-terminal half of GRP94 can account for the peptide binding activity of the full-length protein, ... More
Smooth muscle calponin-caltropin interaction: effect on biological activity and stability of calponin.
AuthorsWills FL, McCubbin WD, Kay CM
JournalBiochemistry
PubMed ID8180179
'Calponin inhibits actomyosin Mg2+ ATPase and is proposed to regulate smooth muscle contraction; however, the mechanism by which it exerts its effect and the regulation of its behavior is still under investigation. The proposed methods by which calponin regulation is effected include reversible phosphorylation of calponin which would allow contraction ... More
Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor.
AuthorsHarikumar KG, Pinon DI, Wessels WS, Prendergast FG, Miller LJ
JournalJ Biol Chem
PubMed ID11893747
'Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa(488), nitrobenzoxadiazolyl, or acrylodan) ... More
Quantification of the interactions among fatty acid, lysophosphatidylcholine, calcium, dimyristoylphosphatidylcholine vesicles, and phospholipase A2.
AuthorsBent ED, Bell JD
JournalBiochim Biophys Acta
PubMed ID7857976
'The rate of hydrolysis of phosphatidylcholine bilayers by soluble phospholipase A2 (PLA2) is greatly enhanced by the presence in the bilayer of a threshold mole fraction of the reaction products: fatty acid and lysophosphatidylcholine (lyso-PC). The threshold requirement of these products appears to vary as a function of vesicle and ... More
Unique effects of different fatty acid species on the physical properties of the torpedo acetylcholine receptor membrane.
AuthorsAntollini SS, Barrantes FJ
JournalJ Biol Chem
PubMed ID11682474
'To study the effects produced by free fatty acids (FFA) on the biophysical properties of Torpedo marmorata nicotinic acetylcholine receptor-rich native membranes and to investigate the topology of their binding site(s), fluorescence measurements were carried out using the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino) naphthalene) and ADIFAB, an Acrylodan-derivatized intestinal fatty acid-binding ... More
Staphylococcal alpha-toxin: the role of the N-terminus in formation of the heptameric pore -- a fluorescence study.
AuthorsValeva A, Pongs J, Bhakdi S, Palmer M
JournalBiochim Biophys Acta
PubMed ID9168153
'Staphylococcus aureus alpha-toxin forms heptameric pores on eukaryotic cell membranes. Assembly of the heptamer precedes formation of the transmembrane pore. The latter event depends on a conformational change that drives a centrally located stretch of 15 amino acid residues into the lipid bilayer. A second region of the molecule that ... More
Accessibility of the fluorescent reporter group in native, silica-adsorbed, and covalently attached acrylodan-labeled serum albumins.
AuthorsIngersoll CM, Jordan JD, Bright FV
JournalAnal Chem
PubMed ID8797379
'Fluorescence quenching techniques are used to investigate the accessibility of a model biorecognition element-reporter group system when in buffer, surface-adsorbed, and covalently attached to a silica surface. The site-selective fluorescent reporter group, 6-acryloyl(dimethylamino)naphthalene (acrylodan, Ac), is attached covalently (at cysteine-34) to bovine and human serum albumin (BSA and HSA, respectively) ... More
Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase.
AuthorsEswaran D, Ali MS, Shenoy BC, Korotchkina LG, Roche TE, Patel MS
JournalBiochim Biophys Acta
PubMed ID7578224
'We have modified bovine pyruvate dehydrogenase (E1), the first catalytic component of the pyruvate dehydrogenase complex, with pyreneglyoxal. Treatment of E1 with pyreneglyoxal resulted in the loss of enzyme activity. Pyruvate plus thiamin pyrophosphate (TPP) afforded approximately 80% protection against this inactivation and protected two arginine residues per mol of ... More
CacyBP/SIP, a calcyclin and Siah-1-interacting protein, binds EF-hand proteins of the S100 family.
AuthorsFilipek A, Jastrzebska B, Nowotny M, Kuznicki J
JournalJ Biol Chem
PubMed ID12042313
'Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating beta-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915-926). In murine brain, CacyBP/SIP is expressed at a high level, but ... More
Interactions of surfactant protein D with fatty acids.
AuthorsDeSilva NS, Ofek I, Crouch EC
JournalAm J Respir Cell Mol Biol
PubMed ID12816736
'Surfactant Protein D (SP-D) plays important roles in antimicrobial host defense, inflammatory and immune regulation, and pulmonary surfactant homeostasis. The best-characterized endogenous ligand is phosphatidylinositol; however, this lipid interaction at least in part involves the carbohydrate moiety. In this study we observed that SP-D binds specifically to saturated, unsaturated, and ... More
The elongation of yeast prion fibers involves separable steps of association and conversion.
AuthorsScheibel T, Bloom J, Lindquist SL
JournalProc Natl Acad Sci U S A
PubMed ID14983002
'A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion [PSI+], a protein-based genetic element of Saccharomyces cerevisiae. In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of ... More
Development of a fluorescent-tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.
AuthorsSimard JR, Getlik M, Grütter C, Pawar V, Wulfert S, Rabiller M, Rauh D,
JournalJ Am Chem Soc
PubMed ID19572644
'Kinase disregulation disrupts the intricate network of intracellular signaling pathways and contributes to the onset of diseases such as cancer. Although several kinase inhibitors are on the market, inhibitor selectivity and drug resistance mutations persist as fundamental challenges in the development of effective long-term treatments. Chemical entities binding to less ... More
Fluorescent peptide probes for high-throughput measurement of protein phosphatases.
AuthorsNoble JE, Ganju P, Cass AE
JournalAnal Chem
PubMed ID12720338
'A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore ... More
Biomolecular mimicry in the actin cytoskeleton: mechanisms underlying the cytotoxicity of kabiramide C and related macrolides.
AuthorsTanaka J, Yan Y, Choi J, Bai J, Klenchin VA, Rayment I, Marriott G
JournalProc Natl Acad Sci U S A
PubMed ID14612571
'This study characterizes the interactions between kabiramide C (KabC) and related macrolides and actin and establishes the mechanisms that underlie their inhibition of actin filament dynamics and cytotoxicity. The G-actin-KabC complex is formed through a two-step binding reaction and is extremely stable and long-lived. Competition-binding studies show that KabC binds ... More
Genetically engineered binding proteins as biosensors for fermentation and cell culture.
AuthorsGe X, Tolosa L, Simpson J, Rao G
JournalBiotechnol Bioeng
PubMed ID14595785
'The signal-transduction properties and the potential applications of two engineered binding proteins from E. coli were extensively studied. Both proteins have a single cysteine mutation in their polypeptide chains, which allow the introduction of an environmentally sensitive fluorophore: ANS for glucose-binding protein (GBP) and acrylodan for glutamine-binding protein (QBP). Both ... More
A mechanism for calmodulin (CaM) trapping by CaM-kinase II defined by a family of CaM-binding peptides.
AuthorsWaxham MN, Tsai AL, Putkey JA
JournalJ Biol Chem
PubMed ID9651352
'Autophosphorylation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) induces a striking >1,000-fold increase in its affinity for CaM, which has been called CaM trapping. Two peptides modeled after the CaM binding domain of CaM-kinase II were previously shown to kinetically resemble CaM binding to phosphorylated and dephosphorylated forms of ... More
Contribution of factor VIIIa A2 and A3-C1-C2 subunits to the affinity for factor IXa in factor Xase.
AuthorsJenkins PV, Dill JL, Zhou Q, Fay PJ
JournalBiochemistry
PubMed ID15109268
'Contributions of factor (F) VIIIa subunits to cofactor association with FIXa were evaluated. Steady-state fluorescence resonance energy transfer using an acrylodan-labeled A3-C1-C2 subunit and fluorescein-Phe-Phe-Arg-FIXa yielded K(d) values of 52 +/- 10 and 197 +/- 55 nM in the presence and absence of phospholipid vesicles, respectively. A3-C1-C2 was an effective ... More
Bimodal distribution and fluorescence response of environment-sensitive probes in lipid bilayers.
AuthorsKlymchenko AS, Duportail G, Demchenko AP, Mély Y
JournalBiophys J
PubMed ID15111409
'A remarkable heterogeneity is often observed in the spectroscopic properties of environment-sensitive fluorescence probes in phospholipid bilayers. To explain its origin, we provided a detailed investigation of the fluorescence excitation and emission spectra of 4''-dimethylamino-3-hydroxyflavone (probe F) in bilayer vesicles with the variations of fatty acid composition, polar heads, temperature, ... More
Dual-labeled glucose binding protein for ratiometric measurements of glucose.
AuthorsGe X, Tolosa L, Rao G
JournalAnal Chem
PubMed ID14987097
'Highly sensitive glucose monitoring has potential applications in conditions where the glucose levels are below the detection limit of currently available technology. Examples include bioprocess monitoring of bacterial cultures and measurement of minute amounts of human interstitial fluid extracted by iontophoresis. Here we describe a ratiometric glucose sensor capable of ... More
Effect of cholesterol/phospholipid ratio on stimulatory GTP-binding protein function.
AuthorsBai L, Youguo H
JournalBiochem Mol Biol Int
PubMed ID9762414
'The effect of different cholesterol/phospholipid (C/P) ratios on the coupling function between stimulatory GTP-binding protein(Gs) and adenylyl cyclase (AC) in proteoliposomes, and its relationship to the conformational change of Gs were investigated. The results showed that Gs activities of both binding GTP gamma S and stimulating adenylyl cyclase were the ... More
Characterization of calponin binding to actin.
AuthorsLu FW, Freedman MV, Chalovich JM
JournalBiochemistry
PubMed ID7547921
'Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding ... More
The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors.
'Rational protein design is an emerging approach for testing general theories of structure and function. The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value. Here we use the design approach to test the current understanding of the structural principles of allosteric interactions ... More
A continuous fluorescence assay for protein kinase C.
AuthorsMcIlroy BK, Walters JD, Johnson JD
JournalAnal Biochem
PubMed ID1888011
'A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation ... More
Rate and mechanism of the assembly of tropomyosin with actin filaments.
AuthorsWeigt C, Wegner A, Koch MH
JournalBiochemistry
PubMed ID1931989
'The rate of assembly of tropomyosin with actin filaments was measured by stopped-flow experiments. Binding of tropomyosin to actin filaments was followed by the change of the fluorescence intensity of a (dimethylamino)naphthalene label covalently linked to tropomyosin and by synchrotron radiation X-ray solution scattering. Under the experimental conditions (2 mM ... More
Kinetics of molecular chaperone action.
AuthorsSchmid D, Baici A, Gehring H, Christen P
JournalScience
PubMed ID8310296
'Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by ... More
S-100 protein, but not calmodulin, binds to the glial fibrillary acidic protein and inhibits its polymerization in a Ca(2+)-dependent manner.
AuthorsBianchi R, Giambanco I, Donato R
JournalJ Biol Chem
PubMed ID8509402
'S-100 protein, a Ca(2+)-binding protein of the EF-hand type, interacts with the glial fibrillary acidic protein (GFAP) in a Ca(2+)-dependent manner. The binding of S-100 protein to GFAP was investigated by fluorescence spectroscopy using acrylodan-S-100 protein and cross-linking experiments using the bifunctional cross-linker, disuccinimidyl suberate. The binding affinity was observed ... More
Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions.
AuthorsWang J, Gambhir A, Hangyás-Mihályné G, Murray D, Golebiewska U, McLaughlin S
JournalJ Biol Chem
PubMed ID12097325
'A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic ... More
Current progress on esterases: from molecular structure to function.
AuthorsSatoh T, Taylor P, Bosron WF, Sanghani SP, Hosokawa M, La Du BN
JournalDrug Metab Dispos
PubMed ID11950776
This article reports on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 2001 Experimental Biology meeting. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene ... More
Phase-fluorometry study on dielectric relaxation of acrylodan-labeled human serum albumin.
AuthorsBuzády A, Erostyák J, Somogyi B
JournalBiophys Chem
PubMed ID11744192
Dielectric relaxation (DR) of acrylodan-labeled human serum albumin (HSA/AC) was studied by phase-fluorometry. A non-monoexponential behavior of both the total fluorescence--and the DR decays has been found. The protein environment of the fluorescent marker shows DR times ranging from the pico to nanosecond timescale. In fluorescence emission decays measured on ... More
Sulfhydryl-selective fluorescence labeling of lipoprotein(a) reveals evidence for one single disulfide linkage between apoproteins(a) and B-100.
AuthorsSommer A, Gorges R, Kostner GM, Paltauf F, Hermetter A
JournalBiochemistry
PubMed ID1835655
Human lipoprotein(a) and low-density lipoprotein were labeled with two different sulfhydryl-selective fluorescence markers. The hydrophilic fluorophore lucifer yellow iodoacetamide and the apolar compound 6-acryloyl-2-(dimethylamino)naphthalene were used to derivatize free -SH groups in the lipoproteins. Three sulfhydryls could be detected in low-density lipoprotein, whereas only two cysteines were available in lipoprotein(a). ... More
Interaction of acrylodan with human serum albumin. A fluorescence spectroscopic study.
AuthorsMoreno F, Cortijo M, González-Jiménez J
JournalPhotochem Photobiol
PubMed ID10568165
The binding of the fluorescent probe acrylodan (AC) to human serum albumin (HSA) was studied by fluorescence spectroscopy. The binding isotherms could be fitted to two types of sites. Competition experiments using iodoacetamide suggested that AC binds tightly on HSA by the cysteine-34. Attempts were made to find the location ... More
Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.
AuthorsPing ZA, Butterfiel DA
JournalBiophys J
PubMed ID1657229
A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was ... More
Spectral characterization of environment-sensitive adducts of interleukin-1 beta.
AuthorsEpps DE, Yem AW, Fisher JF, McGee JE, Paslay JW, Deibel MR
JournalJ Biol Chem
PubMed ID1531338
We have determined the fluorescence properties of two covalently attached acrylodan derivatives of recombinant human interleukin-1 beta, namely the Cys-8 and Lys-103 adducts. The emission and excitation maxima indicated the presence of two operationally distinct conformers for each probe. The iodide quenching and the kinetics of fluorescence changes associated with ... More
A fluorescent derivative of the oligomycin-sensitivity conferring protein (acrylodan-OSCP). Evidence for polarity changes in the environment of CYS118 of OSCP upon binding to mitochondrial F1.
AuthorsDupuis A, Duszynski J, Vignais PV
JournalBiochem Biophys Res Commun
PubMed ID2880585
The fluorescent probe, 6-acryloyl-2-dimethylaminonaphtalene (acrylodan) was reacted with the oligomycin-sensitivity conferring protein (OSCP). Acrylodan bound covalently to the single cysteinyl residue of the protein. Acrylodan-OSCP was fully competent in conferring oligomycin sensitivity to the mitochondrial F0-F1 ATPase complex. The fluorescence emission peak of acrylodan-OSCP was blue-shifted compared to that of ... More
Synthesis, spectral properties, and use of 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan). A thiol-selective, polarity-sensitive fluorescent probe.
AuthorsPrendergast FG, Meyer M, Carlson GL, Iida S, Potter JD
JournalJ Biol Chem
PubMed ID6408077
Fluorophores processing a 6-acyl-2-dimethylaminonaphthalene moiety show fluorescence that is extremely sensitive to solvent polarity (Weber, G., and Farris, F. J. (1979) Biochemistry 18, 3075-3078). We have synthesized and characterized 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan) which selectively labels thiol moieties in proteins. The quantum yield of this agent is markedly enhanced after reaction with ... More
Dynamics surrounding Cys-34 in native, chemically denatured, and silica-adsorbed bovine serum albumin.
AuthorsWang R, Sun S, Bekos EJ, Bright FV
JournalAnal Chem
PubMed ID7864387
We report the steady-state and time-resolved fluorescence of 6-acryloyl(dimethylamino)naphthalene (acrylodan) covalently attached to Cys-34 in bovine serum albumin (BSA). For this conceptually simple system, complicated fluorescence intensity and anisotropy decay kinetics are observed. The steady-state and time-resolved results demonstrate the presence of an excited-state reaction for the BSA-acrylodan system. Additional ... More
Antiparallel dimer and actin assembly.
AuthorsGrintsevich EE, Phillips M, Pavlov D, Phan M, Reisler E, Muhlrad A,
JournalBiochemistry
PubMed ID20361759
The antiparallel dimer (APD) is a unique actin species, which can be detected in the early stages of actin polymerization. In this work, we introduce novel tools for examination of the effects of the APD on actin polymerization. We document that bifunctional methanothiosulfonate (MTS) reagents are an attractive alternative to ... More
Cholecystokinin-octapeptide affects the fluorescence signal of a single pancreatic acinar cell loaded with the acrylodan-labelled MARCKS peptide, a protein kinase C substrate.
AuthorsNgezahayo A, Lang F, Kolb HA
JournalPflugers Arch
PubMed ID7603834
We used a fluorescent derivative of the myristoylated, alanine-rich C kinase substrate (MARCKS) peptide as a probe for protein kinase C (PKC) activation by cholecystokinin-octapeptide (CCK-8) in isolated pancreatic acinar cell pairs. The diffusion of the acrylodan-labelled MARCKS-peptide into the cell interior could be monitored by the increase of fluorescence ... More
A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein.
AuthorsHuang Y, Fan G, Yang F
JournalBiosci Rep
PubMed ID7849240
Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca2+ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca2+ gradient similar to physiological condition ... More
Mechanism of the targeting action of DnaJ in the DnaK molecular chaperone system.
AuthorsHan W, Christen P
JournalJ Biol Chem
PubMed ID12654915
In the DnaK (Hsp70) molecular chaperone system of Escherichia coli, the substrate polypeptide is fed into the chaperone cycle by association with the fast-binding, ATP-liganded form of the DnaK. The substrate binding properties of DnaK are controlled by its two cochaperones DnaJ (Hsp40) and GrpE. DnaJ stimulates the hydrolysis of ... More
A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity.
AuthorsMandal AK, Bhattacharyya A, Bhattacharyya S, Bhattacharyya T, Roy S
JournalProtein Sci
PubMed ID9568911
Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many ... More
D-Peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system.
AuthorsBischofberger P, Han W, Feifel B, Schönfeld HJ, Christen P
JournalJ Biol Chem
PubMed ID12637539
DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the gamma-phosphate cleavage of DnaK-bound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured ... More
Mechanical and chemical properties of cysteine-modified kinesin molecules.
AuthorsIwatani S, Iwane AH, Higuchi H, Ishii Y, Yanagida T
JournalBiochemistry
PubMed ID10441125
To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase ... More
Calpactin I binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments in a Ca(2+)-dependent manner: implications for concerted regulatory effects of calpactin I and S100 protein on glial filaments.
AuthorsBianchi R, Garbuglia M, Verzini M, Giambanco I, Donato R
JournalBiochim Biophys Acta
PubMed ID7918671
Calpactin I, a heterotetrameric, cytoskeletal protein complex composed of two copies of annexin II cross-linked by two copies of p11, an S100-like protein, binds to the glial fibrillary acidic protein (GFAP) and cosediments with glial filaments (GF) in a Ca(2+)-dependent manner, apparently without affecting GFAP polymerization under the present experimental ... More
Engineering the maltose binding protein for reagentless fluorescence sensing.
AuthorsGilardi G, Zhou LQ, Hibbert L, Cass AE
JournalAnal Chem
PubMed ID7802263
This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine residue using site-directed mutagenesis. The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and ... More
Effect of lesions on the dynamics of DNA on the picosecond and nanosecond timescales using a polarity sensitive probe.
AuthorsSomoza MM, Andreatta D, Murphy CJ, Coleman RS, Berg MA
JournalNucleic Acids Res
PubMed ID15131253
This paper explores the effects of structural modifications on the fast dynamics of DNA and the ability of time-resolved Stokes shift spectroscopy to measure those changes. The time-resolved Stokes shift of a synthetic coumarin base-pair replacement within an oligomer is measured between 40 ps and 40 ns. Comparisons are made ... More
Refolding of urea-denatured tubulin: recovery of nativelike structure and colchicine binding activity from partly unfolded states.
AuthorsGuha S, Bhattacharyya B
JournalBiochemistry
PubMed ID9341209
Tubulin unfolding in urea proceeds via the formation of a partially unfolded intermediate state, stable in 2 M urea, that unfolds further in higher urea concentrations. The intermediate state had spectroscopic properties reminiscent of a molten globule and negligible colchicine binding activity. Refolding of totally unfolded tubulin in 8 M ... More
H-Ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate.
AuthorsHightower KE, Huang CC, Casey PJ, Fierke CA
JournalBiochemistry
PubMed ID9799520
The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the alkylation of a cysteine residue of protein substrates with a 15 carbon farnesyl group. We have developed fluorescence assays to directly measure the affinity of the enzyme for peptide and protein (Ras) substrates. A peptide corresponding to the carboxyl terminus of H-Ras ... More
Coupled responses of the regions near cysteine-190 and the carboxy terminus of rabbit cardiac tropomyosin: fluorescence and circular dichroism studies.
AuthorsClark ID, Burtnick LD
JournalBiochemistry
PubMed ID2271683
Rabbit cardiac tropomyosin was labeled at Cys-190 with either N-(1-pyrenyl)iodoacetamide (Py) or 6-acryloyl-2-(dimethylamino)naphthalene (AD, acrylodan). Half of the labeled sample then was treated with carboxypeptidase A to produce an identically labeled nonpolymerizable form of tropomyosin, NPTM. Investigation of temperature-dependent changes in pyrene excimer emission, acrylodan fluorescence polarization, and tyrosyl circular ... More
Unfolding of acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods.
Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain ... More
Fluorescence resonance energy transfer mapping of subunit delta in spinach chloroplast F1 ATPase.
AuthorsEngelbrecht S, Giakas E, Marx O, Lill H
JournalEur J Biochem
PubMed ID9523699
Despite the considerable progress in the field of F0F1-ATPases caused by solving the 2.8-A structure of mitochondrial F1 ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621-628], little is known about the position and function of the enzyme's small subunits which ... More
Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis.
AuthorsPromdonkoy B, Ellar DJ
JournalBiochem J
PubMed ID12795638
Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for ... More
Role of the C-terminal tail region in the self-assembly of lambda-repressor.
AuthorsBandyopadhyay S, Banik U, Bhattacharyya B, Mandal NC, Roy S
JournalBiochemistry
PubMed ID7711028
Acrylamide quenching of the tryptophan fluorescence of the lambda-repressor at different protein concentrations indicates that one of the three tryptophan residues, W129, W142, and W230, undergoes a change in environment upon self-assembly, from dimer to associated species. Quenching data suggest that this tryptophan residue is inaccessible to low concentrations of ... More
Fluorescence studies on the Ca2+ and Zn2+ binding properties of the alpha-subunit of bovine brain S-100a protein.
AuthorsLeung IK, Mani RS, Kay CM
JournalFEBS Lett
PubMed ID3569515
The single cysteine on the alpha-subunit of bovine brain S-100a protein has been modified with the thiol specific probe, Acrylodan. When the labelled apoprotein was excited at 380 nm the fluorescence emission maximum was centered at 484 +/- 2 nm, suggesting that the probe is in a fairly hydrophobic environment. ... More
Unfolding domains in smooth muscle myosin rod.
AuthorsKing L, Seidel JC, Lehrer SS
JournalBiochemistry
PubMed ID7756308
Gizzard smooth muscle myosin rod, an alpha-helical coiled coil, exhibits two cooperative thermal or denaturant-induced helix unfolding transitions in solutions containing 0.6 M NaCl at neutral pH, when monitored by circular dichroism at 222 nm. The first smaller transition unfolds part of the subfragment 2 (S2) domain, and the main ... More
Half-of-the-sites reactivity of F235C lambda-repressor: implications for the structure of the whole repressor.
AuthorsBandyopadhyay S, Deb S, Bose S, Roy S
JournalProtein Eng
PubMed ID12034859
A site-directed mutation, F235C, was created at the penultimate residue of the lambda-repressor. Measurement of dimer-monomer dissociation constant suggested that dimer-monomer dissociation of the mutant repressor is similar to that of the wild-type. Affinity towards a single operator O(R)1 is also similar to that of the wild-type repressor. The mutant ... 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
Role of the C-terminal extension in the interaction of S100A1 with GFAP, tubulin, the S100A1- and S100B-inhibitory peptide, TRTK-12, and a peptide derived from p53, and the S100A1 inhibitory effect on GFAP polymerization.
AuthorsGarbuglia M, Verzini M, Rustandi RR, Osterloh D, Weber DJ, Gerke V, Donato R
JournalBiochem Biophys Res Commun
PubMed ID9920729
Whereas native and recombinant S100A1 inhibited GFAP assembly, a truncated S100A1 lacking the last six C-terminal residues (Phe88-Ser93) (S100A1Delta88-93) proved unable to do so. The inhibitory effects of native and recombinant S100A1 on GFAP assembly were blocked by both TRTK-12, a synthetic peptide derived from the alpha-subunit of the actin ... More
Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges.
Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence ... More
Structural insights into ligand interactions at the acetylcholinesterase peripheral anionic site.
AuthorsBourne Y, Taylor P, Radic Z, Marchot P
JournalEMBO J
PubMed ID12505979
The peripheral anionic site on acetylcholinesterase (AChE), located at the active center gorge entry, encompasses overlapping binding sites for allosteric activators and inhibitors; yet, the molecular mechanisms coupling this site to the active center at the gorge base to modulate catalysis remain unclear. The peripheral site has also been proposed ... More
Strong interaction between caldesmon and calponin.
AuthorsGraceffa P, Adam LP, Morgan KG
JournalJ Biol Chem
PubMed ID8939993
Caldesmon was labeled at either Cys-153 in the NH2-terminal domain or Cys-580 in the COOH-terminal domain with a 6-acryloyl-2-dimethylaminonaphthalene (acrylodan) fluorescence probe. The addition of smooth muscle calponin to Cys-580-labeled caldesmon resulted in an 18% drop in fluorescence intensity, which titrated with a stoichiometry of 0.9 and a binding constant ... More
Effect of polymerization on the subdomain 3/4 loop of yeast actin.
AuthorsMusib R, Wang G, Geng L, Rubenstein PA
JournalJ Biol Chem
PubMed ID11959868
The Holmes F-actin model predicts a polymerization-dependent conformation change of a subdomain 3/4 loop with a hydrophobic tip (residues 266-269), allowing interaction with a hydrophobic surface on the opposing strand of the filament producing filament stabilization. We introduced cysteines in place of Val(266), Leu(267), and Leu(269) in yeast actin to ... More
Acrylodan can label amino as well as sulfhydryl groups: results with low-density lipoprotein, lipoprotein[a], and lipid-free proteins.
Human plasma lipoprotein[a] and autologous low-density lipoprotein were reacted with the fluorescent probe 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) previously reported to be specific for sulfhydryl groups. Reaction kinetics were biphasic in both cases. The reaction of bovine serum albumin with acrylodan was also biphasic. Monophasic kinetics were observed when protein free sulfhydryl groups ... More